Methods, Devices, Kits and Compositions For Detecting Roundworm, Whipworm and Hookworm

ABSTRACT

Methods, devices, kits and compositions for detecting the presence or absence of one or more helminthic coproantigens in a sample are disclosed herein. The methods, devices, kits and compositions of the present invention may be used to confirm the presence or absence of roundworm, whipworm and/or hookworm in a fecal sample from a mammal and may also be able to distinguish between one or more helminth infections. Confirmation of the presence or absence of roundworm, whipworm and/or hookworm in the mammal may be made, for example, for the purpose of selecting an optimal course of treating the mammal and/or for the purpose of determining whether the mammal has been rid of the infection after treatment has been initiated.

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No.14/694,217, filed Apr. 23, 2015, which is a divisional of U.S. patentapplication Ser. No. 13/110,487, filed May 18, 2011, now U.S. Pat. No.9,040,245, issued May 26, 2015, which is a divisional of U.S. patentapplication Ser. No. 12/467,826, filed May 18, 2009 (now U.S. Pat. No.7,951,547, issued May 31, 2011) which in turn claims priority to U.S.Provisional Patent Applications Ser. Nos. 61/122,260, filed Dec. 12,2008; 61/128,077, filed May 19, 2008; 61/128,079, filed May 19, 2008;61/128,076, filed May 19, 2008; 61/128,099, filed May 19, 2008; and61/122,254, filed Dec. 12, 2008; and is a continuation-in-part of U.S.patent application Ser. No. 11/763,592, filed Jun. 15, 2007 (now U.S.Pat. No. 7,736,660, issued Jun. 15, 2010) and is a continuation-in-partof U.S. patent application Ser. No. 11/763,583, filed Jun. 15, 2007, nowU.S. Pat. No. 9,063,129, issued Jun. 23, 2015, which are incorporated byreference herein in their entirety.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to compositions, devices, kits and methodsfor the detection of and distinguishing between roundworm, whipworm andhookworm in mammals. More particularly, the present invention relates toantibodies and antibody compositions, devices, kits, and methods fordetecting the presence or absence of roundworm antigen, whipworm antigenand hookworm antigen in a sample from a mammal and for distinguishingbetween roundworm, whipworm and hookworm antigens.

2. Description of the Prior Art

Parasitic worm (helminth) infections are common in animals and, if notdiagnosed and treated, can cause serious disease or death. Currentmethods for diagnosis of parasitic worm infections primarily involvemicroscopic examination of fecal samples, either directly in fecalsmears or following concentration of ova and parasites by flotation indensity media. Despite this procedure's high adoption, the method hassignificant shortcomings. These microscopic methods are time consumingand require specialized equipment. In addition, the accuracy of resultsof these methods is highly dependent upon the skill and expertise of theoperator. For example, the presence of whipworms is determined bylooking for eggs, but these are excreted intermittently and in smallnumbers. Hookworms are difficult for the average practitioner to detecteither early in infection or in young animals. The specificity ofroundworm diagnosis using microscopic examination is approximately 50%.

Stool handling is disagreeable and hazardous. Sanitary and inoffensiveprocedures for processing stool are awkward and often complex. Suchprocedures may include weighing, centrifuging and storing, and aredifficult except in a clinical laboratory equipped with a suitableapparatus, protective equipment, and a skilled technician. Therefore,any reduction in the number of steps required to perform a fecal testand any reduction in contact between test operator and the test materialis desirable. Clinical laboratories have been using the immunoassaymethods for the detection of various viruses, bacteria and non-helminthparasites and organisms in feces. However, there remains a need for asimple immunoassay method for the detection of a parasitic worminfection in feces, whole blood or in serum.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a device for specifically bindingand isolating helminthic antigens from a sample, for examplecoproantigens from a fecal sample, the device comprising a solidsupport, wherein the solid support has immobilized thereon at least twoantibodies selected from the group consisting of (a) a first antibodycapable of specifically binding a roundworm coproantigen, but not awhipworm or hookworm coproantigen; (b) a second antibody capable ofspecifically binding a whipworm coproantigen, but not a roundworm orhookworm coproantigen; and (c) a third antibody capable of specificallybinding a hookworm coproantigen, but not a whipworm or roundwormcoproantigen. The device, may be, but is not limited to being, forexample, an ELISA device, such as a lateral flow immunoassay device ormicrotiterplate device. Samples that may be tested for roundworm,whipworm and hookworm by the device include, but are not limited tobeing, feces, digestive tract mucous, urine, whole blood, serum, mammarymilk and whole tissue, such as tissue from mammary gland, intestine,liver, heart, lung, esophagus, brain, muscle, and eye, for example. Thedevice further may include, but need not include, one or more reagentsfor the detection of one or more of the group consisting of: one or morenon-worm parasites, one or more viruses, one or more fungi, and one ormore bacteria.

In yet another aspect, the invention provides a method of detecting thepresence or absence of one or more helminthic antigens in a sample, forexample coproantigens from a fecal sample, the method comprising: (a)contacting a sample from a mammal with at least two antibodies selectedfrom the group consisting of: (i) a first antibody capable ofspecifically binding a roundworm coproantigen, but not a whipworm orhookworm coproantigen; (ii) a second antibody capable of specificallybinding a whipworm coproantigen, but not a roundworm or hookwormcoproantigen; and (iii) a third antibody capable of specifically bindinga hookworm coproantigen, but not a whipworm or roundworm coproantigen(b) forming antibody -coproantigen complexes in the presence of thecoproantigens, if any, in the sample; and (c) detecting the presence orabsence of the antibody-coproantigen complexes, if any. The one or morehelminthic coproantigens include coproantigens of roundworm, such asToxocara canis (T. canis), Toxocara cati (T. cati), Toxocara vitulorum(T. vitulorum), Toxascaris leonina (T. leonina), Baylisascaris procyonis(B. procyonis), Ascaridia galli (A. galli), Parascaris equorum (P.equorum), Ascaris suum (A. suum), or Ascaris lumbricoides (A.lumbricoides), Anisakis simplex (A. simplex), or Pseudoterranovadecipiens (P. decipiens), whipworm such as Trichuris vulpis, Trichuriscampanula, Trichuris serrata, Trichuris suis, Trichuris trichiura,Trichuris discolor, and hookworm, such as Ancylostoma caninum,Ancylostoma braziliense, Ancylostoma duodenal, Ancylostoma ceylanicum,Ancylostoma tubaeforme and Ancylostoma pluridentatum, Necatoramericanus, and Uncinaria stenocephala, for example, in a sampleobtained from a mammal, such as a canine, feline, porcine, bovine, orhuman and distinguishing between roundworm, whipworm and hookworm. Inone aspect, the method is carried out to test a fecal mammalian samplefor roundworm coproantigen, whipworm coproantigen, and hookwormcoproantigen. The method, however, is not limited to being carried outto test a fecal sample. In addition to feces, the sample therefore maybe, but is not limited to being whole blood, serum, mammary milk andwhole tissue, such as tissue from mammary gland, intestine, liver,heart, lung, esophagus, brain, muscle, and eye, for example.

In yet another aspect, the invention provides a method of diagnosingwhether a mammal is infected with one or more parasitic worms, themethod comprising the steps of: (a) contacting a sample from a mammalwith at least two antibodies selected from the group consisting of: (i)a first antibody capable of specifically binding a roundwormcoproantigen, but not a whipworm or hookworm coproantigen; (ii) a secondantibody capable of specifically binding a whipworm coproantigen, butnot a roundworm or hookworm coproantigen; and (iii) a third antibodycapable of specifically binding a hookworm coproantigen, but not awhipworm or roundworm coproantigen (b) forming antibody-coproantigencomplexes in the presence of the coproantigens, if any, in the sample;(c) detecting the presence or absence of the antibody-coproantigencomplexes, if any; and (d) diagnosing the mammal as having: (i) aroundworm infection if a roundworm antibody-coproantigen complex ispresent; (ii) a whipworm infection if a whipworm antibody-coproantigencomplex is present; and (iii) a hookworm infection if a hookwormantibody-coproantigen complex is present. The method may also be used totest for and distinguish between environmental contamination withroundworm, whipworm and/or hookworm. Environmental samples that may betested for roundworm, whipworm and/or hookworm by the device include,but are not limited to soil, decomposing material, or fecal matter fromresidential settings including yards, gardens, sand boxes, playgrounds.Testing locations may also include parks, beaches, forests, farms, orother locations exposed to fecal material from dogs, cats, or othermammalian hosts of roundworms. Feces from indoor and outdoor litterboxes may also be tested.

In yet another aspect, the present invention includes a kit for carryingout one or more steps of the method of the invention. The kit mayoptionally include, for example, the device and one or more of thecompositions of the present invention and instructions for carrying outthe method of the present invention. The kit may further optionallyinclude, for example, one or more indicator reagents, one or moreantibody labeling compounds, one or more antibodies, one or more antigencapture reagents, one or more inhibitors, and one or more wash reagentsto be used as part of the device and/or to be used in carrying out themethod.

In yet another aspect, the present invention includes a device forspecifically binding helminthic antigens from a sample, for examplecoproantigens from a fecal sample, the device comprising a solidsupport, wherein the solid support has immobilized thereon at least twoantibodies selected from the group consisting of: (a) a first antibodycapable of specifically binding a roundworm coproantigen, but not awhipworm or hookworm coproantigen; (b) a second antibody capable ofspecifically binding a whipworm coproantigen, but not a roundworm orhookworm coproantigen; and (c) a third antibody capable of specificallybinding a hookworm coproantigen, but not a whipworm or roundwormcoproantigen; and (d) one or more types of roundworm antigen, whipwormantigen, and/or hookworm antigen, wherein the one or more types ofroundworm antigen, whipworm antigen, and hookworm antigen arespecifically bound to the antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of an ELISA assay, which was carried out byusing a lateral flow device and which tested fecal samples from caninesinfected with roundworm, whipworm and/or hookworm by following themethod of the present invention in a first Example.

FIG. 2 shows the results of an ELISA assay, which was carried out byusing a microtiter plate and which tested fecal samples from caninesinfected with either roundworm, hookworm, whipworm or heartworm byfollowing the method of the present invention in a second Example

FIG. 3 shows the nucleotide sequence of a 1210-nucleotide cDNA sequencefrom whole adult Trichuris vulpis (SEQ ID NO:1).

FIG. 4 shows the nucleotide sequence of a 1059-nucleotide cDNA sequencefrom whole adult Trichuris vulpis. (SEQ ID NO:2).

FIG. 5 shows a comparison alignment of SEQ ID NO:3 and SEQ ID NO:4. Theconsensus sequence of SEQ ID NO:3 and SEQ ID NO:4 is shown as SEQ IDNO:9.

FIG. 6 shows the nucleotide sequence of an 865-nucleotide cDNA sequencefrom whole adult Toxocara canis. (SEQ ID NO:10).

FIG. 7 shows the nucleotide sequence of a 632-nucleotide cDNA sequencefrom whole adult Toxocara cati. (SEQ ID NO:11).

FIG. 8 shows a comparison alignment of SEQ ID NO:13 and SEQ ID NO:14.The consensus sequence of SEQ ID NO:13 and SEQ ID NO:14 is shown as SEQID NO:16.

FIG. 9 shows the nucleotide sequence of a 535-nucleotide cDNA sequencefrom whole adult Toxocara canis. (SEQ ID NO:17).

FIG. 10 shows the nucleotide sequence of a 536-nucleotide cDNA sequencefrom whole adult Toxocara cati. (SEQ ID NO:18).

FIG. 11 shows a comparison alignment of SEQ ID NO:20 and SEQ ID NO:21.The consensus sequence of SEQ ID NO:20 and SEQ ID NO:21 is shown as SEQID NO:23.

FIG. 12 shows the nucleotide sequence of a 469-nucleotide cDNA sequencefrom whole adult Toxocara canis. (SEQ ID NO:24).

FIG. 13 shows the nucleotide sequence of a 548-nucleotide cDNA sequencefrom whole adult Toxocara cati. (SEQ ID NO:25).

FIG. 14 shows a comparison alignment of SEQ ID NO:27 and SEQ ID NO:28.The consensus sequence of SEQ ID NO:27 and SEQ ID NO:28 is shown as SEQID NO:30.

FIG. 15 shows an ELISA with elution fractions from SP columns as samplesand that Copro6728 can be partially purified and enriched by eluting theSP column by following the method of the present invention in the thirdExample.

FIG. 16 shows that the molecular weight of Copro6728 was about 7 KDusing a western Blot probed with rabbit anti full-length DIV6728 IgG-HRPfollowing the method of the present invention in the third Example.

FIG. 17 shows that the molecular weight of Copro6728 was about 7 KDusing an SDS-PAGE gel stained with Imperial Protein Staining followingthe method of the present invention in the third Example.

FIG. 18. shows the amino acid sequence of the full length DIV6728 (SEQID NO: 21) with the two peptides (SEQ ID NO: 35 and SEQ ID NO: 36)identified by Mass Spectrometry analysis identified by highlighting themin the shaded boxes following the method of the present invention in thethird Example.

FIG. 19 shows an alignment of the 6728N (SEQ ID NO: 37) and 6728C (SEQID NO: 38) amino acid sequences encoded by the constructs following themethod of the present invention in the fourth Example.

FIG. 20 shows a SDS-PAGE gel loaded with different samples to check thepurification of the recombinant 6728N following the method of thepresent invention in the fourth Example.

FIG. 21 shows a SDS-PAGE gel loaded with different samples to check thepurification of the recombinant 6728C following the method of thepresent invention in the fourth Example.

FIG. 22 shows the ELISA data obtained with different fecal samples totest the different polyclonal antibodies against different recombinant6728 proteins following the method of the present invention in thefourth Example.

FIG. 23 shows the ELISA data obtained with recombinant proteins to testthe different polyclonal antibodies against different recombinant 6728proteins following the method of the present invention in the fourthExample.

FIG. 24 shows Western blotting with different fecal samples probed withrabbit anti-full-length DIV6728 IgG-HRP following the method of thepresent invention in the fourth Example.

FIG. 25 shows Western blotting with different fecal samples probed withrabbit anti-6728C IgG-HRP following the method of the present inventionin the fourth Example.

FIG. 26 shows Western blotting with different fecal samples probed withrabbit anti-6728N IgG-HRP following the method of the present inventionin the fourth Example.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

I. Introduction

The present invention is generally directed to methods, devices, andkits for detecting and distinguishing between roundworm, whipworm andhookworm in a fecal sample obtained from a mammal. The present inventionrelates to roundworm coproantigens from Toxocara, such as Toxocara canisor Toxocara cati, whipworm coproantigens from Trichuris, such asTrichuris vulpis, and hookworm coproantigens Ancylostoma such asAnclostoma caninum, for example. In particular, the present inventionrelates methods, devices and kits for detecting and distinguishingbetween roundworm, such as Toxocara, Toxascaris, Baylisascaris,Ascaridia, Parascaris, Ascaris, Anisakis, or Pseudoterranova, includingT. canis, T. cati, T. vitulorum, T. leonina, T. vitulorum, B. procyonis,A. galli, P. equorum, A. lumbricoides, and A. suum, A. simplex, and P.decipiens, whipworm, such as Trichuris vulpis, Trichuris serrata, T.campanula, and Trichuris trichiura, and hookworm, such as Ancylostomacaninum, Ancylostoma braziliense, Ancylostoma duodenal, Ancylostomaceylanicum, Ancylostoma tubaeforme and Ancylostoma pluridentatum,Necator americanus, and Uncinaria stenocephala, for example.

The present invention provides a superior alternative to the existingmicroscopic inspection techniques. This is true because the presentinvention provides devices, kits and methods for detecting the presenceor absence of roundworm, whipworm and hookworm in a sample from a mammalthat: (1) are both easy to use and yield consistently reliable results;(2) allow for the absence or presence of whipworm in a mammal to beconfirmed regardless of whether that mammal is infected with hookworm,roundworm, and/or heartworm; (3) can detect roundworm, whipworm andhookworm prior to the time that the ova first appear in the infectedhost's feces; and (4) can distinguish between roundworm, whipworm andhookworm infections.

The present invention is based in part on the discovery of unexpectedproperties of compositions specific to roundworm, whipworm and hookworminfections. Specifically, it was determined that antibodies raisedagainst worm specific polypeptides (or raised against an extract ofwhole worms, or extract of worm reproductive organs, or extract of wormintestines) can be used to capture, detect, and distinguish betweenroundworm antigens, whipworm antigens, and hookworm antigens in amammal. The specificity for each type of worm is surprising becauseroundworms, whipworms, and hookworms all are related nematodes, and anantibody raised against a protein isolated from any one of these wormswould be expected to crossreact with one or more of the other worms,host antigens, or other host components.

It was further determined that this antibody can be used to capture anddetect roundworm, whipworm and hookworm antigens in a mammal as early as9 days after the mammal is first infected. This ability to detectroundworm, whipworm or hookworm so soon after infection, and before theappearance of any ova in the feces of the infected mammal, is surprisingbecause ova generally do not appear in the feces of an infective hostuntil about five-to-eight weeks after the host becomes infected.

The present invention therefore includes methods, devices, and kits thatuse antibodies and/or fragments thereof to specifically capture anddetect and distinguish between roundworm, whipworm and hookworm antigensin a mammal. The ability of the present invention to detect and diagnoseroundworm even when one or more other worm types are also present allowsthe mammal's caregiver the opportunity to optimally select a treatmentfor ridding the roundworm, whipworm and/or hookworm from the mammal.Further, the ability of the present invention to, in some cases, detectroundworm, whipworm and/or hookworm as early as 9 days after the mammalis first infected provides the possibility that the caregiver may beginsuch treatment before the mammal becomes severely sickened. Anintervention prior to appearance of ova in the feces would also greatlyreduce or eliminate the possibility that the infestation is spread toother animals or humans.

II. Definitions and Uses of Term

The term “compositions of the invention” refers to all of the nucleicacids, polypeptides, antibodies, and mixtures that include one or moreof those nucleic acids, polypeptides, and antibodies and one or moreother compounds, that can be used to detect the presence or absence ofroundworm, whipworm and/or hookworm in a sample obtained from a mammalby carrying out the method of the present invention that are explicitlydescribed, implicitly encompassed or otherwise disclosed herein.

“A sample from a mammal” in which roundworm, whipworm and/or hookwormcan be detected by the present invention includes all bodily componentsand extracts thereof, such as any fluid, solid, cell or tissue, that arecapable of containing roundworm, whipworm and/or hookworm antigen.Exemplary samples therefore include, but are not limited to being,feces, milk, whole blood and portions thereof, including serum, andfurther include tissue extracts, including tissue from mammary gland,intestine, liver, heart, lung, esophagus, brain, muscle, and eye, forexample. The sample may be taken directly from the mammal or the samplemay be taken from anything that has contacted the mammal. For example,the sample may be fresh or decaying fecal droppings from the mammal. Asanother example, the sample may include soil, dirt, sand, plantmaterial, or any other material that may be mixed with bodily componentsthat may be left behind by a mammal, such as feces, for example. Assuch, the sample may be taken from an environmental source, includingsoil, decomposing material, or fecal matter from forests, farms, orresidential settings, including litter boxes, yards, gardens, sandboxes, playgrounds, parks, and beaches. No matter the origin or thecontent of the sample, this sample sometimes is referred to herein asthe “sample”, the “mammalian sample”, the “test sample” or the “sampleunder test”.

As used herein, “nucleic acid” is synonymous with, and therefore is usedinterchangeably with, “gene”, “DNA”, “cDNA”, “EST”, “polynucleotide”,“oligonucleotide”, “polynucleic acid”, “RNA” and “mRNA”. A nucleic acidmay be in double-stranded form or it may be in single-stranded form.Further, a nucleic acid is either naturally isolated, such as from awhole roundworm, whipworm and/or hookworm or a portion thereof, forexample, or it is artificially synthesized, either in a recombinant hostorganism or by any other artificial means known to the skilled artisan,such as by employing a PCR-based technique, by creating a transgenicorganism that synthesizes the nucleic acid, by using a DNA synthesizingmachine, or by any another molecular-based technique, for example.

“Polypeptide”, “peptide” and “protein” are synonymous terms that areused interchangeably herein to refer to a polymer of amino acidresidues. A polypeptide, peptide and protein of the present inventionmay be either naturally isolated, such as from a whole roundworm,whipworm or hookworm or from a portion of roundworm, whipworm orhookworm for example, or artificially synthesized, either in arecombinant host organism or by any other artificial means known to theskilled artisan.

The term “antibody” or “antibody of the present invention” refers to anyantibody that is able to specifically bind to one or more antigens forthe particular worm without binding to antigens from the other worms.For example antibodies to the one or more roundworm antigens are able tospecifically bind to one or more roundworm antigens, but not to anyantigens from hookworm or whipworm and antibodies to the one or morewhipworm antigens are able to specifically bind to one or more whipwormantigens, but not to any antigens from roundworm and hookworm. Theantibodies of the present invention may be raised against one or moreimmunogenic polypeptides of the present invention. Unless otherwisestated, it is to be understood that the antibody of the presentinvention may include a mixture of two or more different types ofantibody. For example, the antibody may be a mixture of two types ofantibodies, wherein one of the two types specifically binds to oneparticular antigen and the other of the two types specifically binds tosome other antigen.

The term “first antibody” as used herein means one or more antibodiescapable of specifically binding a roundworm coproantigen, but not awhipworm or hookworm coproantigen.

The term “second antibody” as used herein means one or more antibodiescapable of specifically binding a whipworm coproantigen, but not aroundworm or hookworm coproantigen.

The term “third antibody” as used herein means one or more antibodiescapable of specifically binding a hookworm coproantigen, but not awhipworm or roundworm coproantigen.

The “immunogenic polypeptide of the present invention” and, more simply,“the polypeptide of the present invention”, is an immunogen againstwhich the antibodies of the present invention may be raised. All“polypeptides of the present invention” are immunogenic and thereforemay be used to elicit an immune response in a host animal to produce theantibodies of the present invention. Unless otherwise stated, it is tobe understood that the polypeptide of the present invention may be onecomponent of a mixed composition of a plurality of components.

An “immunogen” is any agent, such as the immunogenic polypeptide of thepresent invention, for example, that is capable of eliciting an immuneresponse in an animal that is exposed to that agent.

The term “roundworm”, as used herein, refers to helminths such asintestinal roundworms of the order Ascaridida, which includes the generaToxocara, Toxascaris, Baylisascaris, Ascaridia, Parascaris, Ascaris,Anisakis, and Pseudoterranova. Thus, the term “roundworm”, as usedherein, does not refer to the entirety of the phylum Nematoda.Therefore, “roundworm” does not include any member of the generaAncylostoma, Uncinaria, Necator, Trichuris, Wuchereria, Brugia orDirofilaria.

A “roundworm coproantigen” or a “coproantigen of roundworm” is anyroundworm product that is present in the feces of a mammal having aroundworm infection and that may be specifically bound by one or more ofthe antibodies of the invention. For example, a roundworm coproantigenmay be, but is not limited to being, one or more of the polypeptides ofthe invention. The present inventors have determined that a novelC-terminal 7 kD isoform of DIV6728, which is a excretory/secretoryprotein of T. canis, is present in feces of T. canis-infected canines asearly as 38 days after the canines first became infected with the T.canis. Therefore, a “roundworm coproantigen” may be this novelC-terminal 7 kD isoform of DIV6728 (which is referred to herein as“Copro6728”) that has been observed in canine feces by the presentinventors.

The term “whipworm”, as used herein, refers to helminths such asintestinal whipworms of the genera Trichuris and Trichocephalus.Exemplary whipworms therefore include Trichuris vulpis, Trichuriscampanula, Trichuris serrata, Trichuris suis, Trichuris trichiura,Trichuris discolor and Trichocephalus trichiuris. Further, the term“whipworm”, as used herein, does not refer to the entirety of the phylumNematoda. For example, “whipworm” does not include any member of thegenera Ancylostoma, Uncinaria, Necator, Toxocara, Toxascaris, Ascaris,Wuchereria, Brugia or Dirofilaria.

A “whipworm coproantigen” or a “coproantigen of whipworm” is anywhipworm product that is present in the feces of a mammal having awhipworm infection and that may be specifically bound by one or more ofthe antibodies of the invention. For example, a whipworm coproantigenmay be, but is not limited to being, one or more of the polypeptides ofthe invention.

The term “hookworm,” as used herein, refers to helminthes such asintestinal hookworm of the genera Ancylostoma, Necator and Uncinaria.Exemplary hookworms therefore include Ancylostoma caninum, Ancylostomabraziliense, Ancylostoma duodenal, Ancylostoma ceylanicum, Ancylostomatubaeforme and Ancylostoma pluridentatum, Necator americanus, andUncinaria stenocephala. Further, the term “hookworm,” as used herein,does not refer to the entirety of the phylum Nematoda. For example,“hookworm” does not include any member of the genera Trichuris,Trichocephalus Toxocara, Toxascaris, Ascaris, Wuchereria, Brugia orDirofilaria.

A “hookworm coproantigen” or a “coproantigen of hookworm” is anyhookworm product that is present in the feces of a mammal having ahookworm infection and that may be specifically bound by one or more ofthe antibodies of the invention. For example, a hookworm coproantigenmay be, but is not limited to being, one or more of the polypeptides ofthe invention. The present inventors have determined that a novelN-terminal 28 kDa isoform of ASP5, which is a excretory/secretoryprotein of Ancylostoma, is present in feces of Ancylostoma-infectedcanines as early as 9 days after the canines first became infected withthe Ancylostoma. Therefore, a “hookworm coproantigen” may be this novelN-terminal 28 kDa isoform of ASP5 (which is referred to herein as“CoproASP5”) that has been observed in canine feces by the presentinventors.

“Specific for”, “specifically binds”, and “stably binds” means that aparticular composition of the invention, such as an antibody,polypeptide, or oligonucleotide of the present invention, for example,recognizes and binds to one or more other agents with greater affinitythan to at least one other agent. As one example, an antibody of thepresent invention is said to be “specific for”, to “specifically bind”,and to “stably bind” roundworm antigens whenever that antibody is ableto recognize and bind to those roundworm antigens with greater affinitythan to any other antigens from a non-roundworm parasitic worm. Suchbinding specificity can be tested using methodology well known in theart, for example, ELISA or a radioimmunoassay (RIA). Based oninformation observed regarding the binding specificity of a particularcomposition of the invention, the method of the present invention can becarried out under conditions that allow that composition to bind to (andtherefore to allow the detection of such binding to) a particular agentor agents, but not to significantly bind other agents, while thoseconditions are maintained. As one example, the method of the presentinvention can be carried out under conditions that allow an antibody ofthe present invention to bind to (and therefore to allow the detectionof such binding to) one or more roundworm antigens present in aparticular sample, but not significantly to any hookworm or whipwormantigen that may be present in that sample, thereby allowing for thedistinction between roundworm, whipworm and hookworm.

“Detecting roundworm” means detecting one or more roundworm-specificproducts, including one or more of the polypeptides, antibodies andnucleic acids of the present invention, or one or more roundwormantigens, or Copro6728, for example. The presence of one or more suchroundworm products in a sample from a mammal is indicative that themammal has a roundworm infection, regardless of whether any wholeroundworm organism or ovum thereof is also present in that sample.Conversely, the absence of one or more such roundworm products a samplefrom a mammal is indicative that the mammal does not have a roundworminfection.

“Detecting whipworm” means detecting one or more whipworm-specificproducts, including one or more of the polypeptides, antibodies andnucleic acids of the present invention, or one or more whipwormantigens, for example. The presence of one or more such whipwormproducts in a sample from a mammal is indicative that the mammal has awhipworm infection, regardless of whether any whole whipworm organism orovum thereof is also present in that sample. Conversely, the absence ofone or more such whipworm products a sample from a mammal is indicativethat the mammal does not have a whipworm infection.

“Detecting hookworm” means detecting one or more hookworm-specificproducts, including one or more of the polypeptides, antibodies andnucleic acids of the present invention, or one or more hookwormantigens, or CoproASP5, for example. The presence of one or more suchhookworm products in a sample from a mammal is indicative that themammal has a hookworm infection, regardless of whether any wholehookworm organism or ovum thereof is also present in that sample.Conversely, the absence of one or more such hookworm products a samplefrom a mammal is indicative that the mammal does not have a hookworminfection.

“Amino acid” refers to naturally occurring and synthetic amino acids.Amino acid residues are abbreviated as follows: Alanine is A or Ala;Arginine is R or Arg; Asparagine is N or Asn; Aspartic Acid is D or Asp;Cysteine is C or Cys; Glutamic Acid is E or Glu; Glutamine is Q or Gln;Glycine is G or Gly; Histidine is H or His; Isoleucine is I or Ile;Leucine is L or Leu; Lysine is K or Lys; Methionine is M or Met;Phenylalanine is F or Phe; Proline is P or Pro; Serine is S or Ser;Threonine is T or Thr; Tryptophan is W or Trp; Tyrosine is Y or Tyr; andValine is V or Val. Except where defined otherwise herein, X or Xaarepresents any amino acid. Other relevant amino acids include, but arenot limited to being, 4-hydroxyproline and 5-hydroxylysine. In allcases, the amino acid sequence of a polypeptide described or otherwisereferred to herein is presented in conventional form in that theleft-most, or first, amino acid residue of the sequence is theN-terminal residue and the right-most, or last, amino acid residue ofthe sequence is the C-terminal residue.

A “conservative variant” of any particular nucleic acid sequenceincludes any sequence having one or more degenerate codon substitutionsto that particular nucleic acid sequence, any sequence having one ormore nucleotide substitutions to, insertions to, and deletions from thatparticular nucleic acid sequence, and the complementary sequence of thatparticular nucleic acid and the conservative variants of thatcomplementary sequence. Conservative variants of a particular nucleicacid sequence preferably have at least about 85% identity, morepreferably have at least about 90% identity, and even more preferably atleast about 95-99% identity, to that particular nucleic acid sequence.Conservative variants of a particular nucleic acid sequence may beartificially synthesized or they may be isolated in their natural formfrom an organism.

A “conservative variant” of any particular polypeptide sequence is anypolypeptide having an amino acid sequence that varies from the aminoacid sequence of that particular polypeptide but still retains thespecific binding properties of that particular polypeptide, such that anantibody of the present invention that is raised against the particularpolypeptide is capable of specifically binding the variant polypeptide.Therefore, for example, a conservative variant of a particularpolypeptide may have one or more amino acid substitutions, deletions,additions, and insertions to that particular polypeptide. For example, aconserved variant of a particular polypeptide may have 30 or fewer, 25or fewer, 20 or fewer, 15 or fewer, 10 or fewer, or 5 or fewer,conserved amino acid substitutions to that particular polypeptide.Conservative variants of a particular polypeptide preferably, but notessentially, have at least about 80% identity, more preferably have atleast about 90% identity, and even more preferably at least about 91-99%identity, to that particular polypeptide. A percent identity for anysubject nucleic acid or amino acid sequence (e.g., any of polypeptidesdescribed herein) relative to another “target” nucleic acid or aminoacid sequence can be determined as follows. First, a target nucleic acidor amino acid sequence of the invention can be compared and aligned to asubject nucleic acid or amino acid sequence, using the BLAST 2 Sequences(Bl2seq) program from the stand-alone version of BLASTZ containingBLASTN and BLASTP (e.g., version 2.0.14). The stand-alone version ofBLASTZ can be obtained at www.ncbi.nlm.nih.gov. Instructions explaininghow to use BLASTZ, and specifically the Bl2seq program, can be found inthe ‘readme’ file accompanying BLASTZ. The programs also are describedin detail by Karlin et al. (1990) Proc. Natl. Acad. Sci. 87:2264; Karlinet al. (1990) Proc. Natl. Acad. Sci. 90:5873; and Altschul et al. (1997)Nucl. Acids Res. 25:3389.

“CoproASP5” refers to an N-terminal 28kD fragment of ASP5 found inmammalian feces.

“Copro6728” refers to a C-terminal 7kD portion of DIV6728 found inmammalian feces In a specific embodiment, copro6728 does not include theC-terminus of the full length DIV6728.

Bl2seq performs a comparison between the subject sequence and a targetsequence using either the BLASTN (used to compare nucleic acidsequences) or BLASTP (used to compare amino acid sequences) algorithm.Typically, the default parameters of a BLOSUM62 scoring matrix, gapexistence cost of 11 and extension cost of 1, a word size of 3, anexpect value of 10, a per residue cost of 1 and a lambda ratio of 0.85are used when performing amino acid sequence alignments. The output filecontains aligned regions, of homology between the target sequence andthe subject sequence. Once aligned, a length is determined by countingthe number of consecutive nucleotides or amino acid residues (i.e.,excluding gaps) from the target sequence that align with sequence fromthe subject sequence starting with any matched position and ending withany other matched position. A matched position is any position where anidentical nucleotide or amino acid residue is present in both the targetand subject sequence. Gaps of one or more residues can be inserted intoa target or subject sequence to maximize sequence alignments betweenstructurally conserved domains (e.g., α-helices, β-sheets, and loops).

The percent identity over a particular length is determined by countingthe number of matched positions over that particular length, dividingthat number by the length and multiplying the resulting value by 100.For example, if (i) a 500 amino acid target sequence is compared to asubject amino acid sequence, (ii) the Bl2seq program presents 200 aminoacids from the target sequence aligned with a region of the subjectsequence where the first and last amino acids of that 200 amino acidregion are matches, and (iii) the number of matches over those 200aligned amino acids is 180, then the 500 amino acid target sequencecontains a length of 200 and a sequence identity over that length of 90%(i.e., 180/200×100=90). It will be appreciated that a nucleic acid oramino acid target sequence that aligns with a subject sequence canresult in many different lengths with each length having its own percentidentity. It is noted that the percent identity value can be rounded tothe nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 isrounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 isrounded up to 78.2. It is also noted that the length value will alwaysbe an integer.

Conservative variants of a particular polypeptide sequence may beartificially synthesized or they may be isolated in their natural formfrom an organism, including from a roundworm organism, such as Toxocaracanis, Toxocara cati, and Ascaris, from a whipworm organism, such asTrichuris and Trichocephalus, and hookworm organism, such asAncylostoma, Necator and Uncinaria for example. In one specific, non-limiting example for roundworm, the polypeptide of the invention havingan amino acid sequence corresponding to SEQ ID NO: 27 shown below is aconservative variant of the polypeptide of the present invention havingan amino acid sequence corresponding to SEQ ID NO:26 in that SEQ IDNO:27 is more than 95% identical to SEQ ID NO:26 over an alignment of126 amino acids. More generally, each one of SEQ ID NO:26, SEQ ID NO:27,SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30 are conserved variants ofeach other. It is also to be understood that other conserved variants ofthe SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ IDNO:30 are contemplated by the present invention as described herein, butthe skilled artisan would recognize that all of these contemplatedvariants are too numerous to list. The skilled artisan will alsorecognize that these variants include, but are not limited to, thosehave one or more substitutions of basic amino acid residues, one or moresubstitutions of acidic amino acid residues, one or more substitutionsof polar amino acid residues, one or more substitutions of hydrophobicamino acid residues, one or more substitutions of aromatic amino acidresidues, and one or more substitutions of small amino acid residues.(“Basic” amino acid residues are K, R and H. “Acidic” amino acidresidues are D and E. “Polar” amino acid residues are N and Q.“Hydrophobic” amino acids are I, L, and V. “Aromatic” amino acidresidues are F, Y, and W. “Small” amino acids are G, S, A, T and M.)

III. Nucleic Acids and Polypeptides of the Invention

The nucleic acids and polypeptides of the invention are described indetail in Provisional Applications: “Methods, Devices, Kits AndCompositions For Detecting Whipworm,” Application Ser. No. 61/128,077,filed May 19, 2008; “Methods, Devices, Kits And Compositions ForDetecting Roundworm,” Application Ser. No. 61/128,079, filed May 19,2008; “Methods, Devices, Kits And Compositions For Detecting Roundworm,”Application Ser. No. 61/128,076, files May 19, 2008; “Methods, Devices,Kits And Compositions For Detecting Roundworm,” Application Ser. No.61/128,099, filed May 19, 2008; “Compositions, Devices, Kits and Methodsfor Detecting Hookworm”, Application Ser. No. 61/122,254, filed Dec. 12,2008; and Utility Applications: “Roundworm Coproantigen Detection”,application Ser. No. 11/763,592, filed Jun. 15, 2007 and “Device, Kitand Method for Hookworm Antigen Detection”, application Ser. No.11/763,583, filed Jun. 15, 2007, “Methods, Devices, Kits, Compositionsfor Detecting Roundworm” filed concurrently herewith, and are allincorporated by reference in their entirety.

In an attempt to identify compositions that may be used to confirm thepresence or absence of roundworm in a fecal sample and to distinguishroundworm from other parasitic worm infections, a plurality ofoligonucleotide primers were designed, synthesized and used in 5′ RACE,3′RACE and RT-PCR reactions that included total RNA isolated from eitherwhole adult Toxocara canis or whole adult Toxocara cati. As a result ofthese efforts, an 469-nucleotide cDNA sequence was deduced from Toxocaracanis (identified herein as SEQ ID NO:25), and a 548-nucleotide cDNAsequence was deduced from Toxocara cati (identified herein as SEQ IDNO:26). (BLAST searches that were carried out using SEQ ID NO:25 and SEQID NO:26 indicated these sequences are likely to encode members ofserine protease inhibitor families that were first identified inAscaris, but that had not been identified in either T. canis or T. catiuntil now.)

In an attempt to identify compositions that may be used to confirm thepresence or absence of whipworm in a fecal sample and to distinguishwhipworm from other parasitic worm infections, a plurality ofoligonucleotide primers were designed, synthesized and used in 5′ RACE,3′RACE and RT-PCR reactions that included total RNA isolated from wholeadult Trichuris vulpis. As a result of these efforts, a 1210-nucleotidecDNA sequence and a 1059-nucleotide cDNA sequence were deduced(identified herein as SEQ ID NO:1 and SEQ ID NO:2, respectively. (BLASTsearches that were carried out using SEQ ID NO:1 and SEQ ID NO:2indicated these sequences are likely to encode porin, which is a majorwhipworm excretory/secretory protein that has been described in themouse parasite Trichuris muris and the human parasite Trichuristrichiuria, but that has not been identified in Trichuris vulpis untilnow.)

Previously, Zhan and colleagues described the molecular identificationand partial characterization of ASP-5, which is an excretory/secretoryprotein of Ancylostoma (See Zhan et al., International Journal forParasitology 33:897-907 (2003)). In their studies, the Zhan groupdescribed a single form of the ASP-5 protein having a mass of about 56kDa, secreted from in vitro cultured parasites.

The ASP-5 protein including an N-terminal His6 tag (SEQ ID NO:33) may beencoded by the nucleic sequence of SEQ ID NO:31:

(SEQ ID NO: 31) ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCGAATTCGAGCTCACCACTTGTCCAGGAAATGATCTAACAGATGCTGAACGCACACTGCTAACTAGGGTGCACAATTCCATTCGACGGGAAATAGCGCAAGGAGTTGCAAACAACTACCATGGTGGTAAACTGCCTGCTGGAAAGAACATATACAGGATGAGATACAGCTGTGAGCTGGAACAGGCTGCTATTGATGCTAGTCAAACCTTCTGTTCCGCATCATTGGAGGAACCACAGAAATATGGACAAAACATCCAAGCATACGTCACACCATCTATAATCGCTCGCCCGAAAAACGACCTTCTTGAAGATGCAGTGAAACAATGGTATCTGCCTGTTATCTACTACGGCCAACGCGACGCGGCCAACAAGTTCACCGATCCGCGCTTGTACACATTTGCAAACCTCGCCTACGACAAGAACACTGCACTTGGCTGTCACTATGCGAAATGTCAAGGCCCTGACAGAATCGTCATTAGTTGCATGTACAACAACGTCGTTCCTGACAACGCTGTGATCTACGAGCCAGGAACTGCTTGCGTAAAAGATCAGGACTGCACTACTTATCCTCAGTCCACATGCAAGGACAGCCTTTGCATTATTCCTACGCCACATCCACCAAATCCACCAAATCCACCACCTGCAATGTGTCCAAACGCTGAAATGACTGATGCAGCACGAAAGAAGGTCCTCGACATGCACAACTGGCGCAGATCGCAGCTCGCTCTGGGAAACGTTCAAAACGGGAAAAATGCTTACAACTGCCCCACTGCAACAGACATGTACAAGATGGAATATGATTGCGACCTCGAGAACAGCGCTCTAGCGTATGCAAAGCAATGTAGTCTCGTTGGTTCAGCAGAAGGAACTCGTCCAGGAGAAGGCGAGAATGTCCACAAAGGCGCTCTCGTAACCGATCCGGAGGCTGCAGTTCAGACCGCAGTTCAAGCATGGTGGAGTCAAATCTCACAAAATGGACTCAATGCACAGATGAAATTCACTGCTTTCTTGAAGGACAAGCCTGACGCTCCGACAGCGTTTACACAGATGGCGTGGGCCAAATCCGTAAAGCTTGGATGTGCTGTCTCTAATTGTCAGGCAGATACCTTCACCGTCTGTAGATACAAAGCTGCCGGAAACATCGTGGGCGAATTCATCTATACCAAGGGAAATGTATGCGACGCCTGTAAAGCCACATGCATTACCGCGGAAGGTCTTTGCCCAACGCCTTGAGCGGCCGC

In an effort to identify tools for capturing and detecting hookwormand/or hookworm antigen in hookworm-infected mammals, the presentinventors have determined that a modified protein of about 28 kDa,rather than the 56 kDa version, is present in the feces of canines thatare infected by Ancylostoma. (This 28 kDa version of ASP5 is referred toherein as “CoproASP5”; the detection of CoproASP5 in feces ofAncylostoma-infected canines is described in the Example sectionincluded herein.) In one aspect, therefore, the present inventionprovides polypeptides that may be used to generate antibodies that maybe used to specifically capture and detect CoproASP5. One suchpolypeptide that may be used to generate antibodies that may be used tobind CoproASP5 is referred to as ASP5-1 polypeptide, which may beencoded by the following nucleic acid sequence:

(SEQ ID NO: 32) ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCGAATTCGAGCTCACCACTTGTCCAGGAAATGATCTAACAGATGCTGAACGCACACTGCTAACTAGGGTGCACAATTCCATTCGACGGGAAATAGCGCAAGGAGTTGCAAACAACTACCATGGTGGTAAACTGCCTGCTGGAAAGAACATATACAGGATGAGATACAGCTGTGAGCTGGAACAGGCTGCTATTGATGCTAGTCAAACCTTCTGTTCCGCATCATTGGAGGAACCACAGAAATATGGACAAAACATCCAAGCATACGTCACACCATCTATAATCGCTCGCCCGAAAAACGACCTTCTTGAAGATGCAGTGAAACAATGGTATCTGCCTGTTATCTACTACGGCCAGCGCGACGCGGCCAACAAGTTTACGGATCCGCGCTTGTACACATTTGCAAACCTCGCCTACGACAAGAACACTGCACTTGGCTGTCACTATGCGAAATGTCAAGGCCCTGACAGAATCGTCATTAGTTGCATGTACAACAACGTCGTTCCTGACAACGCAGTGATCTACGAGCCTGGAACTGCTTGCGTAAAAGATGCGGACTGCACTACTTATCCTCAGTCCACATGCAAGGACAGCCTTTGCATTATTCCTACGCCACATCCACCAAATCCACCAAATCCACCACCAGCAATG AGTCCATGAGCGGCCGC

A skilled artisan will appreciate that due to the degeneracy of thegenetic code, nucleic acid sequences other than SEQ ID NO:32 can codefor the polypeptide of SEQ ID NO:34 if appropriate (silent) codonsubstitutions are made.

Analysis of the roundworm sequences corresponding to SEQ ID NO:24 andSEQ ID NO:25 indicated that each one of these roundworm sequencescontains a large open reading frame (ORF). Specifically, the large ORFof SEQ ID NO:24 corresponds to nucleotides 21 through 446 of SEQ IDNO:24 and is predicted to encode a roundworm polypeptide having thefollowing amino acid sequence:

(SEQ ID NO: 26) MLPITFLLAIIVGAAVAHRKCGPNEEWTECTGCEIKCGQGEQPCPMMCRPPSCECMAGKGLRRTADGRCVPEAQCPKRMVKRDEKCGPNEKFLKCRGCEGTCKERLVPCPRMCKPPGCECPASEGFVRNDKGECIKFDDCPK.

Further, the large ORF of SEQ ID NO:25 corresponds to nucleotides 21through 446 of SEQ ID NO:25 and is predicted to encode a roundwormpolypeptide having the following amino acid sequence:

(SEQ ID NO: 27) MLPLTFLLAFIVGAAVAHRKCGPNEEWTECTGCEMKCGEGETPCPMMCRPPSCECMAGKGLRRTPDGRCVPEAQCPKHMVKRDEKCGKNEKFLKCRGCEGTCKERLVPCPKMCKPPGCECPASEGFVRNDKHECIKFDDCPK.

SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:28, SEQ ID NO: 29 and SEQ ID NO: 30 are outlined in ProvisionalApplication Ser. No. 61/128,076 filed May 19, 2008 which is incorporatedby reference in its entirety.

Analysis of the whipworm sequences corresponding to SEQ ID NO:1 and SEQID NO:2 indicated that each one of these whipworm sequences contains alarge ORF. Specifically, the large ORF of SEQ ID NO:1 corresponds tonucleotides 32 through 1147 of SEQ ID NO:1 and is predicted to encode awhipworm polypeptide having the following amino acid sequence:

(SEQ ID NO: 3) MRLVFHAVIYLTLGFLTDAVREKRGKCPPEPPIAGNTIYCRDDFDCGGRQKCCTIAEGRGCVPPYGEQHFEVVKPGHCPAIPAVTGMANFCNTDGDCDGPKKCCLTSRGYDCTHPLHFPIQPQPPVGQCPPSKPRIPGKWVDICAKHANCPDPEKCCDTEYGNRCMDVGLVPGQGERPGNCPNEPRIRGTKYDCRRDDDCDGVQKCCFTVEGRECVEPSRKPLDKPGHCPPIPADVGSARYCDTDRDCDGPRKCCLSSRGYECKHPVHYPDRVEPLVGECPPSRPRIPGKWVDICSKHANCPDPEKCCDTEYGNRCMDVGLVPGQGEKPANCPKEPRIRGTKYDCRRDDDCDGKQKCCYTTEGRECVHGIWP.

Further, the large ORF of SEQ ID NO:2 corresponds to nucleotides 1through 1059 of SEQ ID NO:2 and is predicted to encode a whipwormpolypeptide having the following amino acid sequence:

(SEQ ID NO: 4) VREKRGKCPPEPPIAGNTIYCRDDFDCGGRQKCCTIAEGRGCVPPYGEQDFEVVKPGHCPAIPAVTGMANFCNTDGDCDGPKKCCLTSRGYDCTHPLHFPIQPQPPVGQCPPSKPRVPGKWVDICAKHANCPDPEKCCDTEYGNRCMDVGLVAGQGERPGNCPNEPRIRGTKYDCRRDDDCDGVQKCCFTVEGRECVEPSRKPLDKPGHCPPIPADVGSARYCDTDRDCDGPRKCCLSSRGYECKHPVHYPDRVEPLVGECPPSRPRIPGKWVDICSKHANCPDPEKCCDTEYGNRCMDVGLVPGQGEKPANCPKEPRIRGTKYDCRRDDDCDGKQKCCYTTEGRECVHG IWP.

SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO; 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are outlinedin Provisional Application Ser. No. 61/128,077 filed May 19, 2008 whichis incorporated by reference in its entirety.

Analysis of the roundworm sequences corresponding to SEQ ID NO:10 andSEQ ID NO:11 indicated that each one of these sequences contains a largeORF. Specifically, as shown in, the large ORF of SEQ ID NO:10corresponds to nucleotides 2 through 616 of SEQ ID NO:11 and ispredicted to encode a roundworm polypeptide having the following aminoacid sequence:

(SEQ ID NO: 12) KKIYGVAASRRRRHHFTLENSLDTHLKWLSHEQKEELLOMKKDGKSKKELQDKIMHYYEHLEGDAKHEATEQLKGGCREILKHVVGEEKAAEIKALKDSGASKDELKAKVEEALHAVTDEEICKQHIAEFGPACKKIYGVAASRRRRHHFTLENSLDTHLKWLSHEQKEELLQMKKDGKSKKELQDKIMHYYEHLEGMLL ALCILY.

Further, the large ORF of SEQ ID NO:11 corresponds to nucleotides 1through 486 of SEQ ID NO:11 and is predicted to encode a roundwormpolypeptide having the following amino acid sequence:

(SEQ ID NO: 13) IYGVAASRRRRHHFTLEKSLDTHLKWLSHEQKEELLKMKKDGKSKKELQDKVMHFYEHLEGDAKHEATEQLKGGCREILKHVVGEEKAAEIKALKDSGASKDELKAKVEDALHAVTDEEKKQHIAEFGPACKEIFGVPIDVRHKRDPYTN MTPDEVAEGLRS.

SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, and SEQ ID NO: 16 are outlined in ProvisionalApplication Ser. No. 61/128,079 filed May 19, 2008 which is incorporatedby reference in its entirety.

Analysis of the roundworm sequences corresponding to SEQ ID NO:17 andSEQ ID NO:18 indicated that each one of these roundworm sequencescontains a large open reading frame (ORF). Specifically, the large ORFof SEQ ID NO:17 corresponds to nucleotides 28 through 456 of SEQ IDNO:17 and is predicted to encode a roundworm polypeptide having thefollowing amino acid sequence:

(SEQ ID NO: 19) MLSVLALFALITFAVAGPESCGPNEVWTECTGCELKCGQDENTPCTLNCRPPSCECSPGRGMRRTNDGRCIPASQCPQHRAKREEQCKPNEQWSPCRGCEGTCAQRFVPCTRNCRPPGCECVAGAGFVRDAEGNCIKFDDCPK.

Further, the large ORF of SEQ ID NO:2 corresponds to nucleotides 29through 457 of SEQ ID NO:18 and is predicted to encode a roundwormpolypeptide having the following amino acid sequence:

(SEQ ID NO: 20) MLSVLALFALITFAVADPKSCGPNEVWTECTGCELKCGQDEDTPCTLNCRPPSCECSPGRGMRRTDDGRCIPASQCPQHRAKREEQCKPNEQWSPCRGCEGTCAQRFVPCTRNCRPPGCECVAGAGFVRDAAGNCIKFDDCPK.

SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO: 22, and SEQ ID NO: 23 are outlined in ProvisionalApplication Ser. No. 61/128,099 filed May 19, 2008 which is incorporatedby reference in its entirety

Analysis of the hookworm sequences corresponding to SEQ ID NO: 31 andSEQ ID NO: 32 indicated that each one of these hookwormworm sequencescontains a large open reading frame (ORF). Specifically, the large ORFof SEQ ID NO:31 is predicted to encode a hookworm polypeptide having thefollowing amino acid sequence:

(SEQ ID NO: 33) MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFELTTCPGNDLTDAERTLLTRVHNSIRREIAQGVANNYHGGKLPAGKNIYRMRYSCELEQAAIDASQTFCSASLEEPQKYGQNIQAYVTPSIIARPKNDLLEDAVKQWYLPVIYYGQRDAANKFTDPRLYTFANLAYDKNTALGCHYAKCQGPDRIVISCMYNNVVPDNAVIYEPGTACVKDQDCTTYPQSTCKDSLCIIPTPHPPNPPNPPPAMCPNAEMTDAARKKVLDMHNWRRSQLALGNVQNGKNAYNCPTATDMYKMEYDCDLENSALAYAKQCSLVGSAEGTRPGEGENVHKGALVTDPEAAVQTAVQAWWSQISQNGLNAQMKFTAFLKDKPDAPTAFTQMAWAKSVKLGCAVSNCQADTFTVCRYKAAGNIVGEFIYTKGNVCDACKATCITAEGLCPTP.The first 38 amino acids of SEQ ID NO:33 are derived from a cloningvector, the skilled artisan will appreciate that this portion may beomitted or substituted with other suitable fusion partners.

Further, the large ORF of SEQ ID NO:32 is predicted to encode a hookwormpolypeptide having the following amino acid sequence:

(SEQ ID NO: 34) MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFELTTCPGNDLTDAERTLLTRVHNSIRREIAQGVANNYHGGKLPAGKNIYRMRYSCELEQAAIDASQTFCSASLEEPQKYGQNIQAYVTPSIIARPKNDLLEDAVKQWYLPVIYYGQRDAANKFTDPRLYTFANLAYDKNTALGCHYAKCQGPDRIVISCMYNNVVPDNAVIYEPGTACVKDADCTTYPQSTCKDSLCIIPTPHPPNPPNPPPAM SP.

The polypeptides of the present invention are encoded for by nucleicacids that have a nucleotide sequence that corresponds to all orportions of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 11, SEQID NO: 17, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 31and SEQ ID NO: 32 and all conservative variants of those sequences. Itis to be understood therefore that the amino acid sequence of thepolypeptide of the present invention is variable.

For example, the polypeptide of the present invention may have an aminoacid sequence that corresponds to all or a portion of SEQ ID NO:3, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9 SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ IDNO: 16, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO:33 and SEQ ID NO: 34 or all or a portion of aconservative variant of SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26,SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO:33and SEQ ID NO: 34.

In one specific example, the roundworm polypeptide of the presentinvention has the following amino acid sequence:

(SEQ ID NO: 28) MHRKCGPNEEWTECTGCEIKCGQGEQPCPMMCRPPSCECMAGKGLRRTADGRCVPEAQCPKRMVKRDEKCGPNEKFLKCRGCEGTCKERLVPCPRMCKPPGCECPASEGFVRNDKGECIKFDDCPK.

In one specific example, the whipworm polypeptide of the presentinvention has the following amino acid sequence:

(SEQ ID NO: 5) MVREKRGKCPPEPPIAGNTIYCRDDFDCGGRQKCCTIAEGRGCVPPYGEQHFEVVKPGHCPAIPAVTGMANFCNTDGDCDGPKKCCLTSRGYDCTHPLHFPIQPQPPVGQCPPSKPRIPGKWVDICAKHANCPDPEKCCDTEYGNRCMDVGLVPGQGERPGNCPNEPRIRGTKYDCRRDDDCDGVQKCCFTVEGRECVEPSRKPLDKPGHCPPIPADVGSARYCDTDRDCDGPRKCCLSSRGYECKHPVHYPDRVEPLVGECPPSRPRIPGKWVDICSKHANCPDPEKCCDTEYGNRCMDVGLVPGQGEKPANCPKEPRIRGTKYDCRRDDDCDGKQKCCYTTEGRECVH GIWP.

In another specific example, the roundworm polypeptide of the presentinvention has the following amino acid sequence:

(SEQ ID NO: 14) MHHFTLENSLDTHLKWLSHEQKEELLQMKKDGKSKKELQDKIMHYYEHLEGDAKHEATEQLKGGCREILKHVVGEEKAAEIKALKDSGASKDELKAKVEEALHAVTDEEKKQHIAEFGPACKKIYGVAAS.

In another specific example, the roundworm polypeptide of the presentinvention has the following amino acid sequence:

(SEQ ID NO: 21) MGPESCGPNEVWTECTGCELKCGQDENTPCTLNCRPPSCECSPGRGMRRTNDGRCIPASQCPQHRAKREEQCKPNEQWSPCRGCEGTCAQRFVPCTRNCRPPGCECVAGAGFVRDAEGNCIKFDDCPK.

In another specific example, the hookworm polypeptide of the inventionincludes an amino acid sequence that is identical to or is homologous toa sequence represented by SEQ ID NO: 34.

The 125 amino acid residues that follow the N-terminal methionineresidue of the roundworm polypeptide corresponding to SEQ ID NO: 28specifically represent the amino acid residues 18 through 142 of SEQ IDNO:26. The N-terminal methionine was artificially added to theN-terminus of this polypeptide by carrying out a standard cloningtechnique. Antibody raised against the polypeptide corresponding to SEQID NO: 28 was useful for detecting roundworm antigen. Because theN-terminal methionine was artificially added, and is not thought tonaturally exist in Toxocara (the residue that is immediately prior tothe histidine residue at position 18 in each one of SEQ ID NO:26 and SEQID NO:27 is alanine, and not methionine), it is therefore contemplatedthat the polypeptide of the present invention may have an amino acidsequence that corresponds to amino acid residues 18 through 142 of SEQID NO:26, or, more specifically:

(SEQ ID NO: 29) HRKCGPNEEWTECTGCEIKCGQGEQPCPMMCRPPSCECMAGKGLRRTADGRCVPEAQCPKRMVKRDEKCGPNEKFLKCRGCEGTCKERLVPCPRMCKPPGCECPASEGFVRNDKGECIKFDDCPK.

The 353 amino acid residues that follow the N-terminal methionineresidue of the whipworm polypeptide corresponding to SEQ ID NO:5specifically represent the amino acid residues 20 through 353 of SEQ IDNO:3. The N-terminal methionine was artificially added to the N-terminusof this polypeptide by carrying out a standard cloning technique.Antibody raised against the polypeptide corresponding to SEQ ID NO:5 wasuseful for detecting whipworm antigen. Because the N-terminal methioninewas artificially added, and is not thought to naturally exist inTrichuris vulpis (the residue that is immediately prior to the valineresidue at position 20 in SEQ ID NO:3 is alanine), it is thereforecontemplated that the polypeptide of the present invention may have anamino acid sequence that corresponds to amino acid residues 20 through353 of SEQ ID NO:3, or, more specifically:

(SEQ ID NO: 6) VREKRGKCPPEPPIAGNTIYCRDDFDCGGRQKCCTIAEGRGCVPPYGEQHFEVVKPGHCPAIPAVTGMANFCNTDGDCDGPKKCCLTSRGYDCTHPLHFPIQPQPPVGQCPPSKPRIPGKWVDICAKHANCPDPEKCCDTEYGNRCMDVGLVPGQGERPGNCPNEPRIRGTKYDCRRDDDCDGVQKCCFTVEGRECVEPSRKPLDKPGHCPPIPADVGSARYCDTDRDCDGPRKCCLSSRGYECKHPVHYPDRVEPLVGECPPSRPRIPGKWVDICSKHANCPDPEKCCDTEYGNRCMDVGLVPGQGEKPANCPKEPRIRGTKYDCRRDDDCDGKQKCCYTTE GRECVHGIWP.

In another specific example, the whipworm polypeptide of the presentinvention has the following amino acid sequence:

(SEQ ID NO: 7) MVREKRGKCPPEPPIAGNTIYCRDDFDCGGRQKCCTIAEGRGCVPPYGEQDFEVVKPGHCPAIPAVTGMANFCNTDGDCDGPKKCCLTSRGYDCTHPLHFPIQPQPPVGQCPPSKPRVPGKWVDICAKHANCPDPEKCCDTEYGNRCMDVGLVAGQGERPGNCPNEPRIRGTKYDCRRDDDCDGVQKCCFTVEGRECVEPSRKPLDKPGHCPPIPADVGSARYCDTDRDCDGPRKCCLSSRGYECKHPVHYPDRVEPLVGECPPSRPRIPGKWVDICSKHANCPDPEKCCDTEYGNRCMDVGLVPGQGEKPANCPKEPRIRGTKYDCRRDDDCDGKQKCCYTT EGRECVHGIWP.

The 353 amino acid residues that follow the N-terminal methionineresidue of the whipworm polypeptide corresponding to SEQ ID NO:7specifically represent the amino acid residues 1 through 353 of SEQ IDNO:4. The N-terminal methionine was artificially added to the N-terminusof this polypeptide by carrying out a standard cloning technique.Antibody raised against the polypeptide corresponding to SEQ ID NO:7 wasuseful for detecting whipworm antigen. Because the N-terminal methioninewas artificially added, it is therefore contemplated that thepolypeptide of the present invention may have an amino acid sequencethat corresponds to amino acid residues 1 through 353 of SEQ ID NO:4,or, more specifically:

(SEQ ID NO: 8) VREKRGKCPPEPPIAGNTIYCRDDFDCGGRQKCCTIAEGRGCVPPYGEQDFEVVKPGHCPAIPAVTGMANFCNTDGDCDGPKKCCLTSRGYDCTHPLHFPIQPQPINGQCPPSKPRVPGKWVDICAKHANCPDPEKCCDTEYGNRCMDVGLVAGQGERPGNCPNEPRIRGTKYDCRRDDDCDGVQKCCFTVEGRECVEPSRKPLDKPGHCPPIPADVGSARYCDTDRDCDGPRKCCLSSRGYECKHPVHYPDRVEPLVGECPPSRPRIPGKWVDICSKHANCPDPEKCCDTEYGNRCMDVGLVPGQGEKPANCPKEPRIRGTKYDCRRDDDCDGKQKCCYTTE GRECVHGIWP.

The 129 amino acid residues that follow the N-terminal methionineresidue of the roundworm polypeptide corresponding to SEQ ID NO: 14specifically represent the amino acid residues 14 through 142 of SEQ IDNO: 12. The N-terminal methionine was artificially added to theN-terminus of this polypeptide by carrying out a standard cloningtechnique. Antibody raised against the polypeptide corresponding to SEQID NO: 14 was useful for detecting roundworm antigen. Because theN-terminal methionine was artificially added, and is not thought tonaturally exist in Toxocara (the residue that is immediately prior tothe histidine residue at position 14 in each one of SEQ ID NO: 12 andSEQ ID NO:13 is arginine, and not methionine), it is thereforecontemplated that the polypeptide of the present invention may have anamino acid sequence that corresponds to amino acid residues 14 through142 of SEQ ID NO: 12, or, more specifically:

(SEQ ID NO: 15) HHFTLENSLDTHLKWLSHEQKEELLQMKKDGKSKKELQDKIMHYYEHLEGDAKHEATEQLKGGCREILKHVVGEEKAAEIKALKDSGASKDELKAKVEEALHAVTDEEKKQHIAEFGPACKKIYGVAAS.

With 128 amino acids, the roundworm protein DIV6728 (SEQ ID NO: 21) isabout 14 kD in size and with a theoretical pI is about 6.54. Thisprotein belongs to TIL superfamily, which is a group of serine proteaseinhibitors. In an effort to identify tools for capturing and detectingroundworm and/or roundworm antigen in roundworm-infected mammals, thepresent inventors have determined that only a truncated portion (about 7kDa) of the full-length (14 kDa) protein, and therefore not the 14 kDaversion, is present in the feces of canines that are infected by T.canis. (This 7 kDa truncated portion of DIV6728 is referred to herein as“Copro6728”; the detection of Copro6728 in feces of T. canis-infectedcanines is described in the Example section included herein.) In oneaspect, therefore, the present invention provides polypeptides that maybe used to generate antibodies that may be used to specifically captureand detect Copro6728.

The 127 amino acid residues that follow the N-terminal methionineresidue of the roundworm polypeptide corresponding to SEQ ID NO: 21specifically represent the amino acid residues 17 through 143 of SEQ IDNO: 19. The N-terminal methionine was artificially added to theN-terminus of this polypeptide by carrying out a standard cloningtechnique. Also as described throughout the Example section, antibodyraised against the polypeptide corresponding to SEQ ID NO: 21 was usefulfor detecting roundworm antigen. Because the N-terminal methionine wasartificially added, and is not thought to naturally exist in Toxocara(the residue that is immediately prior to the glycine residue atposition 17 in each one of SEQ ID NO: 19 and SEQ ID NO: 20 is alanine,and not methionine), it is therefore contemplated that the polypeptideof the present invention may have an amino acid sequence thatcorresponds to amino acid residues 17 through 143 of SEQ ID NO:19, or,more specifically:

(SEQ ID NO: 22) GPESCGPNEVWTECTGCELKCGQDENTPCTLNCRPPSCECSPGRGMRRTNDGRCIPASQCPQHRAKREEQCKPNEQWSPCRGCEGTCAQRFVPCTRNCRPPGCECVAGAGFVRDAEGNCIKFDDCPK.

Further, an alignment of SEQ ID NO: 28 (mostly Toxocara canis-derivedsequence; with the only exception being the N-terminal methionineresidue) to SEQ ID NO: 27 (Toxocara cati-derived sequence) is shown inFIG. 14. Because antibody raised against a polypeptide having sequencecorresponding to SEQ ID NO:28 was useful for detecting Toxocara cati, itis additionally contemplated that the polypeptide of the presentinvention may have the amino acid sequence corresponding to SEQ ID NO:30, wherein the X at position 1 is M or absent, the X at position 2 is Lor absent, the X at position 3 is P or absent, the X at position 4 is Lor absent (or the X at position 4 is I, which occupies position 4 of SEQID NO:26), the X at position 5 is T or absent, the X at position 6 is For absent, the X at position 7 is L or absent, the X at position 8 is Lor absent, the X at position 9 is A or absent, the X at position 10 is For absent (or the X at position 10 is I, which occupies position 10 ofSEQ ID NO:26), the X at position 11 is I or absent, the X at position 12is V or absent, the X at position 13 is G or absent, the X at position14 is A or absent, the X at position 15 is A or absent, the X atposition 16 is V or absent, the X at position 16 is M or A, the X atposition 35 is I or M, the X at position 39 is Q or E, the X at position42 is Q or T, the X at position 65 is A or P, the X at position 78 is Ror H, the X at position 88 is P or K, the X at position 111 is R or K,and the X at position 132 is G or H.

Further, an alignment of SEQ ID NO:3 with respect to SEQ ID NO:4 isshown in FIG. 5. It is additionally contemplated that the polypeptide ofthe present invention may have the amino acid sequence corresponding toSEQ ID NO:9, wherein the X at position 1 is M or absent, the X atposition 2 is R or absent, the X at position 3 is L or absent, the X atposition 4 is V or absent, the X at position 5 is F or absent, the X atposition 6 is H or absent, the X at position 7 is A or absent, the X atposition 8 is V or absent, the X at position 9 is I or absent, the X atposition 10 is Y or absent, the X at position 11 is L or absent, the Xat position 12 is T or absent, the X at position 13 is L or absent, theX at position 14 is G or absent, the X at position 15 is F or absent,the X at position 16 is L or absent, the X at position 17 is T orabsent, the X at position 18 is D or absent, the X at position 19 is Aor is absent (or the X at position 19 is M, which occupies position 19of SEQ ID NO:5), the X at position 69 is H or D, the X at position 136is I or V and the X at position 172 is P or A.

Further, an alignment of SEQ ID NO:14 (mostly Toxocara canis-derivedsequence; with the only exception being the N-terminal methionineresidue) to SEQ ID NO:13 (Toxocara cati-derived sequence) is shown inFIG. 8. Because antibody raised against a polypeptide having sequencecorresponding to SEQ ID NO: 14 was useful for detecting Toxocara cati,it is additionally contemplated that the polypeptide of the presentinvention may have the amino acid sequence corresponding to SEQ IDNO:16, wherein the X at position 1 is I or absent, the X at position 2is Y or absent, the X at position 3 is G or absent, the X at position 4is V or absent, the X at position 5 is A or absent, the X at position 6is A or absent, the X at position 7 is S or absent, the X at position 8is R or absent, the X at position 9 is R or absent, the X at position 10is R or absent, the X at position 11 is R or M, the X at position 18 isN or K, the X at position 37 is Q or K, the X at position 52 is I or V,X at position 55 is Y or F, the X at position 110 is E or D, the X atposition 133 is K or E, the X at position 135 is Y or F, the X atposition 138 is A or P, the X at position 139 is A or I, the X atposition 140 is S or D, the X at position 141 is V or absent, the X atposition 142 is R or absent, the X at position 143 is H or absent, the Xat position 144 is K or absent, the X at position 145 is R or absent,the X at position 146 is D or absent, the X at position 147 is P orabsent, the X at position 148 is Y or absent, the X at position 149 is Tor absent, the X at position 150 is N or absent, the X at position 151is M or absent, the X at position 152 is T or absent, the X at position153 is P or absent, the X at position 154 is D or absent, the X atposition 155 is E or absent, the X at position 156 is V or absent, the Xat position 157 is A or absent, the X at position 158 is E or absent,the X at position 159 is G or absent, the X at position 160 is L orabsent, the X at position 161 is R or absent, and the X at position 162is S or absent.

Further, an alignment of SEQ ID NO: 21 (mostly Toxocara canis-derivedsequence; with the only exception being the N-terminal methionineresidue) to SEQ ID NO: 20 (Toxocara cati-derived sequence) is shown inFIG. 11. Because antibody raised against a polypeptide having sequencecorresponding to SEQ ID NO: 21 was useful for detecting Toxocara cati,it is additionally contemplated that the polypeptide of the presentinvention may have the amino acid sequence corresponding to SEQ IDNO:23, wherein the X at position 1 is absent or M, the X at position 2is absent or L, the X at position 3 is absent or S, the X at position 4is absent or V, the X at position 5 is absent or L, the X at position 6is absent or A, the X at position 7 is absent or L, the X at position 8is absent or F, the X at position 9 is absent or A, the X at position 10is absent or L, the X at position 11 is absent or I, the X at position12 is absent or T, the X at position 13 is absent or F, the X atposition 14 is absent or A, the X at position 15 is absent or V, the Xat position 16 is M or A, the X at position 17 is G or D, the X atposition 19 is E or K, the X at position 42 is N or D, the X at position66 is N or D, and the X at position 132 is E or A.

A polypeptide of the present invention may have the amino acid sequencecorresponding to SEQ ID NO:38, wherein the amino acid at position 54 isE or A. Antibodies that specifically bind to this polypeptide areincluded in the invention.

Because the first 38 amino acid residues of the polypeptide having theamino acid sequence that corresponds to SEQ ID NO: 34 were not derivedfrom Ancylostoma (i.e., they are vector sequence), it is furthercontemplated that the peptide of the present invention may include anamino acid sequence that is identical to or is homologous to a sequencerepresented by SEQ ID NO: 34, wherein the X at position 1 is M orabsent, the X at position 2 is G or absent, the X at position 3 is S orabsent, the X at position 4 is S or absent, the X at position 5 is H orabsent, the X at position 6 is H or absent, the X at position 7 is H orabsent, the X at position 8 is H or absent, the X at position 9 is H orabsent, the X at position 10 is H or absent, the X at position 11 is Sor absent, the X at position 12 is S or absent, the X at position 13 isG or absent, the X at position 14 is L or absent, the X at position 15is V or absent, the X at position 16 is P or absent, the X at position17 is R or absent, the X at position 18 is G or absent, the X atposition 19 is S or absent, the X at position 20 is H or absent, the Xat position 21 is M or absent, the X at position 22 is A or absent, theX at position 23 is S or absent, the X at position 24 is M or absent,the X at position 25 is T or absent, the X at position 26 is G orabsent, the X at position 27 is G or absent, the X at position 28 is Qor absent, the X at position 29 is Q or absent, the X at position 30 isM or absent, the X at position 31 is G or absent, the X at position 32is R or absent, the X at position 33 is G or absent, the X at position34 is S or absent, the X at position 35 is E or absent, the X atposition 36 is F or absent, the X at position 37 is E or absent, and theX at position 38 is L or absent. Furthermore, because the S at position251 in the SEQ ID NO:33 was artificially substituted during the cloningprocess described in the Example section herein in that sequence (theASP5 protein of wild-type Ancylostoma includes a C residue at thatposition), it is contemplated that the X at position 251 of SEQ ID NO:34may be either S or C.

It is also contemplated that any one or more of the SEQ ID NO:3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9 SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ IDNO: 16, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO:33 and SEQ ID NO: 34 may be only a portion of alarger polypeptide sequence, and therefore may represent partialsequence of one or more proteins that normally are expressed inroundworm, for example, or one or more polypeptide sequences that areartificially fused to SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26,SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO:33, SEQ ID NO: 34, or SEQ ID NO: 38, or Copro6728. The skilledartisan will recognize that are a variety of techniques exist forartificially fusing two or more polypeptide fragments together.

It is even further contemplated that the polypeptide of the presentinvention may include more than one of the SEQ ID NO:3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 SEQID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23,SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO:30, SEQ ID NO:33 SEQ ID NO: 34, SEQ ID NO: 38, and Copro6728. Forexample, the polypeptide of the present invention may include the SEQ IDNO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO: 9 SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQID NO: 29, SEQ ID NO: 30, SEQ ID NO:33, SEQ ID NO: 34, or SEQ ID NO: 38,or Copro6728. Also, it is contemplated that the polypeptide of thepresent invention may include a plurality of polypeptide fragmentscorresponding to SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 SEQ ID NO:12, SEQ ID NO:13, SEQID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20,SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO:33, SEQ IDNO: 34, or SEQ ID NO: 38, or Copro6728. For example, the polypeptide ofthe present invention may be formed by a plurality of polypeptidefragments corresponding to SEQ ID NO:5, SEQ ID NO: 14, SEQ ID NO: 21, orSEQ ID NO: 28 that are fused together. In another example, thepolypeptide of the present invention may be formed by a plurality ofpolypeptide fragments corresponding to SEQ ID NO:5 SEQ ID NO: 14, SEQ IDNO: 21, or SEQ ID NO: 28 and a plurality of polypeptide fragmentscorresponding to SEQ ID NO:7, SEQ ID NO: 16, SEQ ID NO: 23, or SEQ IDNO: 30 that are fused together in any combination.

Whereas one particular polypeptide of the present invention wasexpressed and isolated by a specific technique (in which is described inthe Example section included herein), the skilled artisan will recognizethat any of the polypeptides of the present invention may be isolated byemploying any one or more of a variety of techniques. (See, e.g., Sewaldand Jakubke, Peptides: Chemistry and Biology, Wiley Publishing (2002);Peptide Synthesis and Applications (Methods in Molecular Biology) Howl,ed., Humana Press (2005); Jones, Amino Acid and Peptide Synthesis,Oxford University Press (2002), each one of which is incorporated hereinby reference in its entirety.) These techniques include those that maybe carried out to isolate naturally existing polypeptides having aminoacid sequence corresponding to SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 SEQ ID NO:12, SEQID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19,SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO:33, SEQ ID NO: 34, SEQ ID NO: 38, and Copro6728and any naturallyoccurring variant of those polypeptides. These techniques furtherinclude those that may be carried out to artificially generate thepolypeptides having amino acid sequence corresponding to SEQ ID NO:3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9 SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15,SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ IDNO: 29, SEQ ID NO: 30, SEQ ID NO:33, SEQ ID NO: 34, SEQ ID NO: 38, andCopro6728 and any conserved variant of those polypeptides. Such variantsmay be generated, for example, by employing any one or more mutagenesistechniques or by direct synthesis.

The polypeptides of the present invention are capable of eliciting animmune response in a host animal that is exposed to these polypeptidesto produce one or more of the antibodies of the present invention.Regardless of the technique by which they are derived, the polypeptidesof the present invention are preferably prepared in substantially pureform when they are to be used for the purpose of raising antibody.Preferably, these polypeptides are at least about 80% pure, morepreferably are at least about 90-95% pure, and even more preferably areat least about 99% pure. Exemplary techniques for eliciting an immuneresponse in a host organism and for isolating antibodies therefrom aredescribed herein, but it is to be understood that the present inventionis not limited to those techniques. The skilled artisan will recognizethat there are a plurality of techniques for achieving this same goalwithout deviating from the scope and spirit of the invention.

IV. Antibodies of the Invention

The present invention further includes antibodies and antigen-bindingfragments thereof that are raised against and that specifically bind allor part of one or more polypeptides of the present invention, and alsoincludes compositions that include said antibodies and antigen-bindingfragments thereof. When contacted to a sample obtained from a mammal,these antibodies and antigen-binding fragments are able to specificallybind to a particular helminthic worm antigen. For example the roundwormantibodies and antigen-binding fragments are able to specifically bindroundworm antigens present in the sample, but are not able tospecifically bind any antigen from hookworm or whipworm that may bepresent in the sample. As a further example, the whipworm antibodies andantigen-binding fragments are able to specifically bind whipwormantigens present in the sample, but are not able to specifically bindany antigen from hookworm or roundworm that may be present in thesample. The antibodies of the present invention are suitable for beingused only to capture one or more roundworm antigens, whipworm antigensand/or hookworm antigens, only to detect one or more roundworm antigens,whipworm antigens and/or hookworm antigens, or more preferably, to bothcapture and detect one or more roundworm antigens, whipworm antigensand/or hookworm antigens.

The antibodies of the present invention may belong to any antibodyclass, including for example, IgG, IgM, IgA, IgD and IgE, and may beprepared by any of a variety of techniques known to the skilled artisan.(See, e.g., Dean, Methods Mol. Biol. 80:23-37 (1998); Dean, Methods Mol.Biol. 32:361-79 (1994); Baileg, Methods Mol. Biol. 32:381-88 (1994);Gullick, Methods Mol. Biol. 32:389-99 (1994); Drenckhahn et al. MethodsCell. Biol. 37:7-56 (1993); Morrison, Ann. Rev. Immunol. 10:239-65(1992); Wright et al. Crit. Rev. Immunol. 12:125-68(1992); Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory(1988); and Making and Using Antibodies: A Practical Handbook, Howardand Kaser, eds., CRC Press (2006), each one of which is incorporatedherein by reference in its entirety.)

In one technique, the polypeptide of the invention is introduced into ahost animal, such as into rabbit, mouse, rat, guinea pig, goat, pig,cow, sheep, donkey, dog, cat, chicken, or horse, for example. Anenhanced immune response may be elicited in the host animal byassociating the polypeptide with a carrier and/or by exposing the hostto an adjuvant, but it is to be understood that the present inventiondoes not require that the polypeptide be associated with a carrier orthat the host be exposed to the adjuvant. An exemplary carrier that maybe used for this purpose is bovine serum albumin, bovine thyroglobulin,and soybean trypsin inhibitor. Exemplary adjuvants include Freund'scomplete or incomplete adjuvant and MDL-TDM adjuvant. Regardless ofwhether the polypeptide is associated with such a carrier or whether thehost is exposed to an adjuvant, booster immunizations optionally may bemade with the host animal being bled one or more times thereafter.Polyclonal antibodies that specifically bind the polypeptide may then bepurified from antisera obtained from the bleed or bleeds. Suchpurification may be achieved, for example, by employing affinitychromatography techniques that involve associating the polypeptide to asolid support. Such affinity chromatography techniques are well known bythe skilled artisan.

In several embodiments, the roundworm antibody of the present inventionis an antibody that is raised in rabbit by immunizing that host animalwith the polypeptide having the amino acid sequence corresponding to SEQID NO: 28, SEQ ID NO: 14, and SEQ ID NO:21, (Hereinafter, theseparticular antibodies are referred to as “anti-DIV6744,”,“anti-DIV6716,” and “anti-DIV6728 respectively) The skilled artisan willrecognize that the production and isolating of anti-DIV6744,anti-DIV6716 and anti-DIV6728, or any other antibody of the presentinvention, is not limited to any specific technique.

In another embodiment, the roundworm antibody of the present inventionis an antibody that is raised in rabbit by immunizing that host animalwith a polypeptide having the amino acid sequence corresponding to 6728C(SEQ ID NO:38). (Hereinafter, this particular antibody is referred to an“anti-Copro6728C”.) A specific technique for producing and isolatingthis antibody is described in the Example section herein, but theskilled artisan will recognize that the production and isolating ofanti-Copro6728C is not limited to that specific technique.

In additional embodiments, the roundworm antibody of the presentinvention is an antibody that is raised in rabbit by immunizing thathost animal with extract of whole roundworm, extract of roundwormintestine, or extract of roundworm reproductive organs, as described inU.S. application Ser. No. 11/763,592 entitled “Roundworm CoproantigenDetection”, filed Jun. 15, 2007.

In a further embodiment, the whipworm antibody of the present inventionis an antibody that is raised in rabbit by immunizing that host animalwith the polypeptide having the amino acid sequence corresponding to SEQID NO:5 or SEQ ID NO:7 (Hereinafter, this particular antibody isreferred to as “anti-DIV6901” or “anti-DIV6902”.) The skilled artisanwill recognize that the production and isolating of anti-DIV6901,anti-DIV6901 or any other antibody of the present invention, is notlimited to any specific technique.

In another embodiment, a hookworm antibody of the present invention isan antibody that is raised in rabbit by immunizing that host animal withthe polypeptide having the amino acid sequence corresponding to SEQ IDNO: 34. (Hereinafter, this particular antibody is referred to as“anti-Asp5-1”.) The skilled artisan will recognize that the productionand isolating of anti-Asp5-1, or any other antibody of the presentinvention, is not limited to any specific technique.

In an additional embodiment, the hookworm antibody of the presentinvention is an antibody that is raised in rabbit by immunizing thathost animal with the polypeptide having the amino acid sequencecorresponding to SEQ ID NO:33, i.e. with substantially the full-lengthASP5 protein from hookworm, as described in U.S. application Ser. No.11/763,583 entitled “Device, Kit and Method for Hookworm AntigenDetection”, filed Jun. 15, 2007.

In other embodiments, the antibodies of the present invention are raisedin a host against one or more polypeptides having an amino acid sequencethat is a conservative variant of the sequence corresponding to SEQ IDNO: 5, SEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 28, and SEQ ID NO: 34.In some other embodiments, the antibodies of the present invention areraised in a host against any one or more polypeptides having an aminoacid sequence corresponding to the sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ IDNO: 29, SEQ ID NO: 30, SEQ ID NO:33, SEQ ID NO: 38, Copro6728 andCoproAP5, or one or more polypeptides having an amino acid sequence thatis a conservative variant of any of those sequences.

In another embodiment, the antibodies of the present invention areantibodies that specifically bind one or more the polypeptides havingthe amino acid sequence corresponding to SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9 SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30,SEQ ID NO:33, SEQ ID NO: 34, SEQ ID NO: 38, Copro6728 or CoproAP5, orantigenic portions thereof.

In yet other embodiments, the antibodies of the present inventionspecifically bind one or more polypeptides having an amino acid sequencethat is a conservative variant of the sequence corresponding to SEQ IDNO:5, SEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 28, or SEQ ID NO: 34. Insome other embodiments, the antibodies of the present inventionspecifically bind one or more polypeptides having an amino acid sequencecorresponding to the sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9 SEQ ID NO:12, SEQ ID NO:13, SEQID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22,SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO: 38, Copro6728 or CoproAP5, or one or morepolypeptides having an amino acid sequence that is a conservativevariant of any of those sequences.

It is also to be understood that the antibodies of the inventionoptionally may be polyclonal or monoclonal antibodies, single chainantibodies (scFv), chimeric antibodies, and fragments thereof.Monoclonal antibodies that are specific for the polypeptide of interestmay be obtained and purified, for example, by preparing cell lines thatgenerate antibodies having the desired specificity to the polypeptide ofinterest. Cell lines of this kind may be derived from cells of aparticular type (e.g., spleen cells) that are isolated from a hostanimal that had previously been immunized with the polypeptide asdescribed before. In such a case, these cells could then beimmortalized, for example, by fusing them with myeloma cells by carryingout any one of a variety of fusion techniques known to the skilledartisan. In one exemplary technique, the cells from the immunized hostanimal are co-incubated with their fusion partner, e.g., the myelomacells, in the presence of a detergent for a short period of time beforebeing plated on a medium that supports the growth of hybrid cells (butnot the myeloma fusion partner). Such selection may be achieved, forexample, by using hypoxanthine, aminopterin, and thymidine (HAT). Whenhybrid cells emerge during selection, in perhaps one or two weeks aftercommencing the selection process, single hybrid colonies (and theirsupernatants) are tested for their ability to bind the polypeptide orpolypeptides against which the host animal was immunized. Hybridcolonies having the most optimal binding specificity would represent thebest candidates from which monoclonal antibodies may be isolated. Thesemonoclonal antibodies, for example, may be isolated directly from thesupernatant (i.e., medium) in which these colonies are grown byemploying any one of a variety techniques known to the skilled artisan.

The antibodies of the invention also may be a single chain antibody(scFv), or an antigen binding fragment of an antibody. Antigen-bindingfragments of antibodies are a portion of an intact antibody comprisingthe antigen binding site or variable region of an intact antibody,wherein the portion is free of the constant heavy chain domains of theFc region of the intact antibody. Examples of antibody fragments includeFab, Fab′, Fab′-SH, F(ab′)₂ and F_(v) fragments. In addition toproduction and purification from animals or mammalian cells, antibodies,antibody fragments, or non-antibody scaffolds can be selected based uponvarious in vitro technologies, including phage display, ribosomaldisplay, or bacterial display.

Antibodies, including secondary antibodies, may be labeled with any typeof label known in the art, including, for example, fluorescent,chemiluminescent, radioactive, enzymes, colloidal particles,radioisotopes and bioluminescent labels. In various embodiments of theinvention, the one or more of the antibodies of the invention arelabeled with an enzyme, a colloidal particle, a radionuclide or afluorophor. The particulate label can be, for example, a colored latexparticle, dye sol, or gold sol conjugated to an antibody.

Methods, Devices and Kits of the Invention

Devices and Kits of the Invention

The present invention, in one aspect, is a detecting the presence orabsence of one or more helminthic antigens from a sample, the devicecomprising a solid support, wherein the solid support has immobilizedthereon at least two antibodies selected from the group consisting of(a) a first antibody capable of specifically binding a roundwormcoproantigen, but not a whipworm or hookworm coproantigen; (b) a secondantibody capable of specifically binding a whipworm coproantigen, butnot a roundworm or hookworm coproantigen; and (c) a third antibodycapable of specifically binding a hookworm coproantigen, but not awhipworm or roundworm coproantigen; and optionally, (d) one or moretypes of roundworm coproantigen, whipworm coproantigen, and/or hookwormcoproantigen, wherein the one or more types of roundworm coproantigen,whipworm coproantigen, and hookworm coproantigen are specifically boundto the antibodies. The device is arranged to aid specifically bindingand isolating helminthic coproantigens from roundworm, whipworm andhookworm in a sample from a mammal.

In one aspect, the device includes a solid support, wherein one or moreantibodies of the invention are immobilized on the solid support. Thesolid support may be, but is not limited to being, the inner, bottomsurface of a well of a microtiter plate or a substrate that is includedas part of a lateral flow device, for example. An exemplary microtiterplate is an Immulon 1B 96-well plate (which is commercially availablefrom Thermo Scientific of Milford, Mass.), but it is to be understoodthat the skilled artisan will recognize that a large variety of othermicrotiter plates that are not the Immulon 1B 96-well plate allow forthe immobilization of antibodies thereon, and therefore would besuitable for providing the solid support of the present invention.

An exemplary lateral flow device is the lateral flow device that isdescribed in U.S. Pat. No. 5,726,010, which is incorporated herein byreference in its entirety. The device for performing a lateral flowassay may be a SNAP® device, which is commercially available from IDEXXLaboratories, Inc. of Westbrook, Me. However, it is to be understoodthat the skilled artisan will recognize that a large variety of otherlateral flow devices that are not SNAP® devices or described by U.S.Pat. No. 5,726,010 allow for the immobilization of an antibody thereon,and therefore would be suitable for being used as the device of thepresent invention. These devices can include, for example, lateral flowdevices that use colloidal gold technology.

Antibodies used in the device of the invention may be immobilized on thesolid support by any methodology known in the art, including, forexample, covalently or non-covalently, directly or indirectly, attachingthe antibodies to the solid support. Therefore, while these antibodiesmay be attached to the solid support by physical adsorption (i.e.,without the use of chemical linkers), it is also true that theseantibodies may be immobilized to the solid support by any chemicalbinding (i.e., with the use of chemical linkers) method readily known toone of skill in the art.

It is also to be understood that the solid support may be any suitablematerial for the immobilization of the antibodies of the invention. Forexample, the solid support may be beads, particles, tubes, wells,probes, dipsticks, pipette tips, slides, fibers, membranes, papers,natural and modified celluloses, polyacrylamides, agaroses, glass,polypropylene, polyethylene, polystyrene, dextran, nylon, amylases,plastics, magnetite or any other suitable material readily known to oneof skill in the art.

The device optionally may include one or more labeled antigen capturereagents that may be mixed with a sample from a mammal prior toapplication to a device of the invention. When the labeled captureantigen reagent is included, the labeled antigen capture reagent may ormay not be deposited or dried on a solid surface of the device. “Antigencapture reagent” refers to any compound that is specific for the antigenor antigens of interest. The labeled antigen capture reagent, whetheradded to the mammalian sample or pre-deposited on the device, may be,for example, a labeled antibody specific for a roundworm antigen,including, but not limited to, the antibodies of the present invention.In one example, anti-DIV6744 conjugated with horseradish peroxidase maybe used as a labeled antigen capture reagent. In another example,anti-DIV6901 or anti-DIV6902 conjugated with horseradish peroxidase maybe used as a labeled antigen capture reagent. In a further example,anti-DIV6716 conjugated with horseradish peroxidase may be used as alabeled antigen capture reagent. In a further example, anti-DIV6728conjugated with horseradish peroxidase may be used as a labeled antigencapture reagent. In yet a further example, anti-Copro6728 conjugatedwith horseradish peroxidase may be used as a labeled antigen capturereagent.

The device also may optionally include a liquid reagent that transports(such as when the device is a SNAP® device, for example), or otherwisefacilitates removal of (such as when the device includes a microtiterplate, for example), unbound material (e.g., unreacted portions of themammalian sample, such as, for example, unreacted portions of fecalextract, and unbound antigen capture reagent) away from the reactionzone (solid phase). The liquid reagent may be a wash reagent and serveonly to remove unbound material from the reaction zone, or it mayinclude a detector reagent and serve to both remove unbound material andfacilitate antigen detection. For example, in the case of an antigencapture reagent conjugated to an enzyme, the detector reagent includes asubstrate that produces a detectable signal upon reaction with theenzyme-antibody conjugate at the reaction zone (solid phase).Alternatively, in the case of a labeled antigen capture reagentconjugated to a radioactive, fluorescent, or light-absorbing molecule,the liquid reagent acts merely as a wash solution facilitating detectionof complex formation at the reactive zone by washing away unboundlabeled reagent.

The liquid reagent may further include a limited quantity of an“inhibitor”, i.e., a substance that blocks the development of thedetectable end product. A limited quantity is defined as being an amountof inhibitor sufficient to block end product development until most orall excess, unbound material is transported away from the second region,at which time detectable end product is produced.

The device of the present invention may also include various bindingreagents immobilized at locations distinct from the antigen capturereagent or reagents. For example, an immunoreagent (an antibody, antigenor polypeptide) that recognizes a species-specific (e.g.,roundworm-specific) antibody portion of a labeled antibody or antigencapture reagent, or an enzyme portion of an enzyme-labeled reagent, canbe included as a positive control to assess the viability of thereagents within the device. For example, a positive control may be ananti-horseradish peroxidase antibody that has been raised in, forexample, goat or mouse. Additionally, a reagent, e.g., an antibody,isolated from a non-immune member of the species from which the antibodyportion of the antigen-antibody complex was derived can be included as anegative control to assess the specificity of immunocomplex (i.e.,antigen-antibody complex) formation.

In addition to being designed to specifically binding and isolatinghelminthic coproantigens from roundworm, whipworm and hookworm in amammalian sample, the device of the invention optionally may be designedto allow one or more other diagnostic tests to be performed. Forexample, the solid support may also include reagents for the detectionof one or more non-worm parasites, one or more viruses, one or morefungi, or one or more bacteria. The reagents for the detection of one ormore non-worm parasites, one or more viruses, one or more fungi, or oneor more bacteria may be, for example, one or more antibodies or one ormore antigens recognized by antibodies specific for one or more non-wormparasites, one or more viruses, one or more fungi, or one or morebacteria.

In one embodiment, the device of the present invention is a microtiterplate that includes a plurality of wells, wherein each well includes asolid support having anti-DIV6744 pAB immobilized thereupon.

The plate may be used in conjunction with a method of the presentinvention to detecting the presence or absence of one or more helminthiccoproantigens in a sample. For example, a roundworm infection may bediagnosed in a mammal by detecting one or more roundworm antigens withthe anti-DIV6744 pAB, one or more whipworm antigens with theanti-DIV6902 pAB, and one or more hookworm antigens with the anti-Asp5-1pAB, that is immobilized on the solid support. In one embodiment, theantigens that are detected are coproantigens. “Coproantigens” are anyproduct or products of roundworm, whipworm or hookworm that are presentin a fecal sample and that can specifically bind to antibodies.Coproantigens therefore may be whole worm, worm eggs, worm fragments, orproducts secreted, excreted or shed from worm or a combination thereof.Coproantigens further include the polypeptides of the present invention,such as the polypeptides having an amino acid sequence corresponding toSEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQID NO: 8, SEQ ID NO: 9 SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ IDNO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28,SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO:33, SEQ ID NO: 34, or SEQ ID NO:38, polypeptides having an amino acid sequence that is a conservativevariant of those sequences, and/or antigenic fragments of any suchpolypeptides, and CoproASP5 and Copro6728, for example.

The invention further includes assay kits (e.g., articles ofmanufacture) for detecting and distinguishing between roundworm,whipworm and/or hookworm in a mammalian sample. A kit therefore mayinclude one or more devices and/or compositions of the presentinvention. For example, the kit may include anti-roundworm antibodiesand means for determining binding of the antibodies to roundwormantigens, anti-whipworm antibodies and means for determining binding ofthe antibodies to whipworm antigens, and anti-hookworm antibodies andmeans for determining binding of the antibodies to hookworm antigens inthe sample. In one particular example, such a kit includes the devicehaving an immobilized anti-roundworm antibody, such as anti-DIV6744, ananti-whipworm antibody, such as anti-DIV6902, and an anti-hookwormantibody, such as anti-Asp5-1, for example, one or more antigen capturereagents (e.g., a non-immobilized labeled antigen capture reagent and animmobilized antigen capture reagent) and wash reagent, as well asdetector reagent and positive and negative control reagents, if desiredor appropriate. Other components such as buffers, controls, and thelike, known to those of ordinary skill in art, may be included in suchtest kits. The relative amounts of the various reagents can be varied,to provide for concentrations in solution of the reagents thatsubstantially optimize the sensitivity of the assay. Particularly, thereagents can be provided as dry powders, usually lyophilized, which ondissolution will provide for a reagent solution having the appropriateconcentrations for combining with a sample. The present kit may furtherinclude instructions for carrying out one or more methods of the presentinvention, including instructions for using any device and/orcomposition of the present invention that is included with the kit.

B. Methods of the Invention

The present invention further includes methods for using one or more ofthe devices, kits and/or compositions of the present invention to detectthe presence or absence of one or more helminthic antigens in a sample.The methods therefore may be carried out to detect the presence orabsence of roundworm, whipworm and/or hookworm in a sample, such as, forexample, a fecal sample, that is obtained from a mammal, including, butnot limited to, a canine, feline, porcine, bovine or human. Further, themethods may be carried out to detect Toxocara, such as T. canis or T.cati, or T. vitulorum, Ascaris, such as A. lumbricoides or A. suum,Anisakis, such as A. simplex, Pseudoterranova, such as P. decipiens,Trichuris and/or Trichocephalus, such as Trichuris vulpis, Trichuriscampanula, Trichuris serrata, Trichuris suis, Trichuris trichiura,Trichuris discolor and Trichocephalus trichiuris, Ancylostoma caninum,Ancylostoma braziliense, Ancylostoma duodenal, Ancylostoma ceylanicum,Ancylostoma tubaeforme and Ancylostoma pluridentatum, Necatoramericanus, and Uncinaria stenocephala, for example.

In the methods of the present invention, detection of roundworm,whipworm and/or hookworm may be accomplished by detecting the presenceor absence of one or more roundworm, whipworm and/or hookworm antigens,such as Copro6728 and CoproASP5 or the polypeptides having an amino acidsequence corresponding to SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26,SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO:33, SEQ ID NO: 34, or SEQ ID NO: 38 as well as antigenic fragmentsand/or conservative variants of those sequences, and CoproASP5, forexample. When the sample under test for helminthic coproantigens isfeces, the soluble portion of the feces may be collected by any protocolknown in art. For example, in addition to the specific protocoldescribed in the Example section herein, the soluble portions of thesample generally may be collected by using filtration, extraction,centrifugation, or simple mixing followed by gravimetric settling. Theskilled artisan will recognize that there are a variety of ways ofextracting and preparing non-fecal samples from a mammal as well. Forexample, the sample may be a bodily fluid that is naturally excreted orotherwise released by the mammal or that is artificially obtained fromthe mammal. Such artificial extraction may be carried out by milking themammal or by injecting a syringe into the mammal and drawing the fluidinto the syringe. Once obtained, the fluid optionally may befractionated (for example, serum may be fractionated from whole blood asthen used as the sample). As another example, the sample may be obtainedby swabbing the mammal, such as the oral cavity of the mammal, forexample. As yet another example, tissue sections may be obtained bybiopsy.

The methods include contacting the mammalian sample with one or moreantibodies specific for helminthic coproantigens under conditions thatallow an antigen/antibody complex, i.e., an immunocomplex, to form. Thatis, an antibody specifically binds to a coproantigen present in thesample. The skilled artisan is familiar with assays and conditions thatmay be used to detect such antigen/antibody complex binding. Forexample, the antigen/antibody complex may be detected using a secondaryantibody that binds to the antigen/antibody complex. The formation of acomplex between antigen and antibodies in the sample may be detectedusing any suitable method known in the art.

Further, the relative amount of antibody-antigen complexes that areformed in one particular reaction may be measured with respect to thoseformed in any other reaction by any methodology known in the art forachieving that goal. When it is determined that a sample under test hasa specific (roundworm, whipworm and/or hookworm) antibody-antigencomplexes, it can be concluded, based upon the specific complexesformed, that a specific helminth is present in the host mammal and whichhelminth is present (roundworm, whipworm and/or hookworm). When this istrue, it may be concluded that the mammal from which the test sample wasobtained harbors an intestinal helminth infection. The conclusions thatthe mammal being tested harbors an intestinal helminth infection may bemade by a clinician at a diagnostic service provider or by a caregiverof the mammal, such as the mammal's veterinarian, for example. When acaregiver of a mammal determines (or is otherwise informed that) amammal harbors a helminth infection and which helminth is present, thecaregiver may then subject the mammal to a course of treatment that isoptimally designed to rid the mammal of the helminth specifically,rather than of a parasitic nematode infection generally. Further, thepresent invention can be used to confirm that any animal that hasreceived treatment for the specific helminth infection has been rid ofthat infection. A caregiver who learns that a sample includes bothroundworm and whipworm, but not hookworm, for example, could use thatknowledge to treat the mammal from which the sample was takenspecifically for roundworm by administering to that mammal a drugoptimally effective against roundworm and a second drug optimallyeffective against whipworm. Absent such knowledge, the caregiver may,for example, otherwise treat the mammal with a drug that is optimallyeffective against only roundworm, only whipworm, or neither roundwormnor whipworm (in such cases, the mammal would be at risk of receivingsuboptimal treatment). In addition, humans who may come in contact withthe infested animal or its excretions may be advised to take precautionsagainst acquiring the parasite or parasites. In this context, it isimportant to determine the worm species with high specificity, as somehelminths, such as roundworms and hookworms, can cause significantdisease (e.g., larval migrans) in humans, while it is generally acceptedthat whipworm does not play a zoonotic role of importance in humans.

The steps of the method of the present invention may include applying amammalian sample to a device of the invention, which includes a firstantibody capable of specifically binding a roundworm coproantigen, butnot a whipworm or hookworm coproantigen; a second antibody capable ofspecifically binding a whipworm coproantigen, but not a roundworm orhookworm coproantigen; and a third antibody capable of specificallybinding a hookworm coproantigen, but not a whipworm or roundwormcoproantigen to form antibody-coproantigen complexes in the presence ofthe coproantigens, if any, in the sample; and detecting the presence orabsence of the antibody-coproantigen complexes, if any. Antibodiesspecific for antigens of roundworms may be directly or indirectlyattached to a solid support or a substrate such as a microtiter well,antibody-immobilizing portion of a SNAP® device, magnetic bead,non-magnetic bead, column, matrix, membrane, fibrous mat composed ofsynthetic or natural fibers (e.g., glass or cellulose-based materials orthermoplastic polymers, such as, polyethylene, polypropylene, orpolyester), sintered structure composed of particulate materials (e.g.,glass or various thermoplastic polymers), or cast membrane film composedof nitrocellulose, nylon, polysulfone or the like (generally syntheticin nature). All of these substrate materials may be used in suitableshapes, such as films, sheets, or plates, or they may be coated onto orbonded or laminated to appropriate inert carriers, such as paper, glass,plastic films, or fabrics. Suitable methods for immobilizing peptides onsolid phases include ionic, hydrophobic, covalent interactions and thelike.

The methods of the present invention do not require the use of solidphases or substrates, however. The skilled artisan will recognize thatthere are a number of ways that the present method may be carried out todetect the presence or absence of roundworm without involving the use ofsolid phases or substrates. In just one example, immunoprecipitationmethods that do not require the use of solid phases or substrates may becarried out.

In some embodiments of the invention, the antigen/antibody complex isdetected when an indicator reagent, such as an enzyme conjugate, whichis bound to the antibody, catalyzes a detectable reaction. Optionally,an indicator reagent including a signal generating compound may beapplied to the antigen/antibody complex under conditions that allowformation of a detectable antigen/antibody/indicator complex.Optionally, the antibody may be labeled with an indicator reagent priorto the formation of an antigen/antibody complex.

The formation of an antigen/antibody complex or anantigen/antibody/indicator complex in some of the methods of the presentinvention specifically may be detected by radiometric, colorimetric,fluorometric, photometric, size-separation, or precipitation methods.Detection of an antigen/antibody complex also may be accomplished by theaddition of a secondary antibody that is coupled to an indicator reagentincluding a signal generating compound. Indicator reagents includingsignal generating compounds (labels) associated with apolypeptide/antibody complex may be detected using the methods describedabove and may include chromogenic agents, catalysts such as enzymeconjugates, fluorescent compounds such as fluorescein and rhodamine,chemiluminescent compounds such as dioxetanes, acridiniums,phenanthridiniums, ruthenium, and luminol, radioactive elements, directvisual labels, as well as cofactors, inhibitors, magnetic particles, andthe like. Examples of enzyme conjugates include alkaline phosphatase,horseradish peroxidase, beta-galactosidase, and the like. The selectionof a particular label is not critical, but it will be capable ofproducing a signal either by itself or in conjunction with one or moreadditional substances.

Methods of the invention include, but are not limited to those based oncompetition, direct reaction or sandwich-type assays, including, but notlimited to ELISA, RIA, immuno-fluorescent assays (IFA), hemagglutination(HA), fluorescence polarization immunoassay (FPIA), and microtiter plateassays (i.e., any assay done in one or more wells of a microtiterplate). One assay of the invention includes a reversible flowchromatographic binding assay, which may be performed, for example, byusing a SNAP® device. See U.S. Pat. No. 5,726,010.

In some embodiments, the method of the invention facilitates sandwich orcompetition-type specific binding assays. In a sandwich assay, antigencapture reagents are immobilized in a reactive zone. These antigencapture reagents may specifically bind to antigens in the sample beingtested for roundworm, whipworm and/or hookworm. Following binding of theantigen from the sample, the antigen capture reagent/antigen complex isdetected by any suitable method. For example, the complex may be reactedwith labeled specific binding reagents (e.g., an enzyme-antibodyconjugate) and antigen detected (e.g., upon reaction with substrate).

In other embodiments of the method of the present invention, acompetition assay is performed. In a competition assay, antigen capturereagents are immobilized at the reactive zone and are contactedsimultaneously with antigen from a sample and labeled antigen (e.g., anantigen-enzyme conjugate). The amount of label detected at the reactivezone is inversely proportional to the amount of antigen in the sample.

In some embodiments of the method, antibodies specific for a roundworm,whipworm and hookworm coproantigens are attached to a solid phase orsubstrate. A sample potentially including an antigen from roundworm,whipworm and/or hookworm are added to the substrate. Antibodies thatspecifically bind roundworm, whipworm and/or hookworm are added. Theantibodies may be the same antibodies used on the solid phase or theymay be from a different source or species. Further, these antibodies maybe linked to an indicator reagent, such as an enzyme conjugate. Washsteps may be performed prior to each addition. A chromophore or enzymesubstrate may be added and color may be allowed to develop. The colorreaction may be stopped and the color may be quantified using, forexample, a spectrophotometer, and/or the color may be subjectivelyassessed by the human eye.

In other embodiments of the method, antibodies specific for a roundworm,whipworm and hookworm coproantigens are attached to a solid phase orsubstrate. A sample potentially including a roundworm, whipworm and/orhookworm antigen is added to the substrate. Second anti-speciesantibodies that specifically bind the coproantigens are added. Thesesecond antibodies are from a different species than are the solid phaseantibodies. Third anti-species antibodies that specifically bind thesecond antibodies and that do not specifically bind the solid phaseantibodies are added. The third antibodies may include an indicatorreagent, such as an enzyme conjugate. Wash steps may be performed priorto each addition. A chromophore or enzyme substrate may added and colormay be allowed to develop. The color reaction may be stopped and thecolor may be quantified using, for example, a spectrophotometer, and/orthe color may be subjectively assessed by the human eye.

In a specific example, the method of the present invention is performedin conjunction with a device that is a lateral flow assay device byadding a prepared mammalian sample to a flow matrix of the device at afirst region (a sample application zone). The prepared sample is carriedin a fluid flow path by capillary action to a second region of the flowmatrix where a particulate label capable of binding and forming a firstcomplex with an antigen in the sample exists. The particulate label canbe, e.g., a colored latex particle, dye sol, or gold sol conjugated toan antibody specific for a roundworm antigen. The first complex iscarried to a third region of the flow matrix where an antibody thatspecifically binds a roundworm antigen is immobilized at a distinctlocation. A second complex is formed between the immobilized antibodyand the first complex. The particulate label that is part of the secondcomplex can be directly visualized by the human eye.

Each specific helminth antibody may be an immobilized antigen capturereagent in a reaction zone (solid phase). A second antigen capturereagent, i.e., a second specific helminth antibody that has beenconjugated to a label, either may be added to the sample before thesample is added to the device, or the second antigen capture reagent canbe incorporated into the device. For example, the labeled antigencapture reagent may be deposited and dried on a fluid flow path thatprovides fluid communication between a sample application zone and thesolid phase. Contact of the labeled antigen capture reagent with thetest sample can result in dissolution of the labeled antigen capturereagent.

In one embodiment of the method of the present invention, specifichelminthic coproantigen is detected by ELISA. Specific examples of theELISA method of the present invention is described in the Examplesection included herein. Although the present invention is describedwith respect to those specific ELISA methods, however, it is to beunderstood that those of ordinary skill in the art will recognize thatalternative, additional or substitute ELISA steps may be used withoutdeviating from the basic goal achieved through this method of theinvention.

In another embodiment of the present invention, helminthic coproantigenis detected by using a lateral flow device, such as a SNAP® device, forexample.

Further, the methods of the invention for detection of helminthinfection can be combined with other diagnostic assays to detect thepresence of other organisms or conditions. For example, assays of theinvention can be combined with reagents that detect one or more non-wormfecal parasites, one or more viruses, one or more fungi, one or morebacteria, one or more blood-borne parasites or occult blood or acombination thereof. By providing two or more unique binding sites in asingle assay device (such as, for example, two unique spots on a SNAP®assay device), the present invention allows for detection of two or moreorganisms from a single sample. In one embodiment, there are threeunique spots for detection of past or present infection or infestationfrom three organisms (the spots being either antigen or antibody bindingreagents) from a single sample (i.e., the same individual sample isexposed to the three capture reagents on a single device). In yetanother embodiment, there are four unique spots for detection of past orpresent infection or infestation from four organisms (the spots beingeither antigen or antibody binding reagents) from a single sample (i.e.,the same individual sample is exposed to the four capture reagents on asingle device. It is to be understood, however, that the same device mayinclude more than four unique spots and/or allow for the detection ofmore than four organisms.

The reagents for the detection of one or more non-worm parasites, one ormore viruses, one or more fungi, or one or more bacteria may be, forexample, one or more antibodies or one or more antigens recognized byantibodies specific for one or more non-worm parasites, one or moreviruses, one of more fungi, or one or more bacteria.

The method further may optionally include using one or more nucleicacids from roundworm, whipworm and hookworm, including, but not limitedto, the nucleic acids of the present invention, to determine thepresence or absence of roundworm, whipworm and/or hookworm in amammalian sample. Such use of these nucleic acids for determining thepresence of the helminth may be carried out before, after orconcomitantly with the carrying out of any other aspects of the method,including the detection of roundworm, whipworm and hookworm by antibody.Therefore, in one aspect, after roundworm, whipworm and/or hookworm isdetected or not detected in a particular sample and the mammal fromwhich the sample was obtained is diagnosed as either having or nothaving a roundworm, whipworm and/or hookworm infection, the sample (or alater-obtained sample from the diagnosed mammal) may be tested for thepresence or absence of any one or more of the nucleic acids, includingany one or more nucleic acids of the invention. Anyone failing to detecta specific helminth in a particular mammal by using one or more nucleicacids (after the helminth had been detected by using one or moreantibodies) would need to take into consideration the possibility thatthe antibodies had detected helminthic corpoantigen prior to theappearance of detectable helminthic nucleic acid in the sample. In suchan instance, the mammal's caregiver may elect to ignore the observationthat the nucleic acid had failed to detect the helminth and proceed withtreating the mammal specifically for helminth infection based on theobservation that the antibodies had in fact detected helminth. Inanother aspect, the nucleic acids are used to determine the presence orabsence of helminths in a particular mammal, and then the presence orabsence of helminths is further evaluated by using the antibodies of thepresent invention. Detection of one or more helminthic nucleic acids maybe carried out by using any nucleic acid detection techniques known tothe skilled artisan. For example, such detection may be carried out byperforming a PCR-based technique, such as, but limited to, for example,a real-time PCR-based technique. Exemplary PCR-based techniques aredescribed in, e.g., PCR Protocols (Methods in Molecular Biology), 2^(nd)ed., Bartlett and Stirling, eds., Humana Press (2003); and Sambrook andRussell, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press (2001); each one of which is incorporated herein byreference in its entirety.

The present invention is specifically described with reference to fiveExamples; however, it is not to be construed as being limited thereto.

EXAMPLES

Unless otherwise indicated, the following materials and techniques wereused to generate data described in one or more of Examples 1-4 asdescribed below.

Polyclonal roundworm antibody preparation. The polyclonal antibodies“anti-DIV6728 pAB,” (IgG) was raised in rabbit against a polypeptidehaving amino acid sequence corresponding to SEQ ID NO:21, respectivelyand purified from serum by using standard methods. Briefly, foranti-DIV6728 pAB nucleotides 76 through 456 of SEQ ID NO:17 were clonedin-frame into a vector (D8223, which is a derivative of pUC19) to createthe plasmid D8245. Specifically, the 125 amino acids of SEQ ID NO: 21that follow the methionine residue at the N-terminus of that sequencecorrespond to a portion of SEQ ID NO:19 and are encoded for by thecloned portion of SEQ ID NO:17. In the D8245 plasmid, the N-terminalmethionine residue was encoded for by vector sequence at the junction ofthat plasmid where the vector was ligated to the cloned sequence fromSEQ ID NO:17.

DNA sequence encoding SEQ ID NO:21 was then cleaved from the D8245plasmid by restriction exonuclease digestion (NdeI and BamHI) andpurified. This purified sequence was then ligated to linearizedexpression vector, pET28a, and the resulting circular construct(pTDX198::DIV6728) was transformed into BL21 (DE3) E. coli cells. (Thecomplete sequence of the insert was confirmed by DNA sequence analysis.)Expression of His-tagged fusion protein was induced by addition of 1 mMIPTG to cultures of the transformed E. coli. Recombinant protein wassolubilized in 6 M urea and purified by nickel affinity and ion exchangechromatography. (This recombinant protein is hereinafter is referred toas “rDIV6728”.)

After rDIV6728 was introduced into rabbits, anti-DIV6728 pAB waspurified from the plasma of the immunized rabbits by isolating IgGantibody by protein G affinity chromatography.

Polyclonal whipworm antibody preparation. The polyclonal antibody“anti-DIV6901 pAB” (IgG) was raised in rabbit against a polypeptidehaving amino acid sequence corresponding to SEQ ID NO:5 and purifiedfrom serum by using standard methods. Briefly, in the case ofanti-DIV6901 pAB, nucleotides 89 through 1147 of SEQ ID NO:1 were clonedin-frame into an expression vector (D8223, which is a derivative ofpUC19) to create the plasmid D9073. Specifically, the 353 amino acids ofSEQ ID NO:5 that follow the methionine residue at the N-terminus of thatsequence correspond to a portion of SEQ ID NO:3 and are encoded for bythe cloned portion of SEQ ID NO:1.

DNA sequence encoding SEQ ID NO:5 was then cleaved from the D9073plasmid by restriction exonuclease digestion (NdeI and BamHI) andpurified. This purified sequence was then ligated to linearizedexpression vector, pET28a, and the resulting circular construct(ptDX233::DIV6901) was transformed into E. coli cells. (The completesequence of the insert was confirmed by DNA sequence analysis.)Expression of His-tagged fusion protein was induced by addition of 1 mMIPTG to cultures of the transformed E. coli. Recombinant protein wassolubilized in 6 M urea and purified by nickel affinity and ion exchangechromatography. (This recombinant protein is hereinafter is referred toas “rDIV6901”.) Anti-DIV6901 pAB was purified from the plasma of theimmunized rabbits by isolating IgG antibody by protein G affinitychromatography.

Polyclonal hookworm antibody preparation and isolation. The polyclonalantibody anti-ASP5-1 (IgG) was raised in rabbit against a polypeptidehaving amino acid sequence corresponding to SEQ ID NO: 34 and purifiedfrom serum by using standard methods. Briefly, nucleotides 50 through427 of SEQ ID NO: 32 were cloned in-frame into a plasmid. Specifically,the 129 amino acids of SEQ ID NO: 34 that follow the methionine residueat the N-terminus of that sequence correspond to a portion of SEQ ID NO:33 and are encoded for by the cloned portion of SEQ ID NO: 32. In theplasmid, the N-terminal methionine residue was encoded for by vectorsequence at the junction of that plasmid where the vector was ligated tothe cloned sequence from SEQ ID NO: 32.

DNA sequence encoding SEQ ID NO: 32 was then cleaved from the plasmid byrestriction exonuclease digestion (NotI and SacI) and purified. Thispurified sequence was then ligated to linearized expression vector,pET28a, and the resulting circular construct was transformed into BL21(DE3) E. coli cells. (The complete sequence of the insert was confirmedby DNA sequence analysis.) Expression of His-tagged fusion protein wasinduced by addition of 1 mM IPTG to cultures of the transformed E. coli.Recombinant protein was solubilized in 6 M urea and purified by nickelaffinity and ion exchange chromatography. (This recombinant protein ishereinafter is referred to as “rASP5-l”.)

After rASP5-1 was introduced into rabbits, anti-ASP5-1 pAB was purifiedfrom the plasma of the immunized rabbits by isolating IgG antibody byprotein G affinity chromatography.

Infection of canine and feline animals. Parasitic nematode infection waseffected by orally administering about 150-300 larvated eggs ofroundworm (Toxocara canis), 150-300 infective larvae of hookworm(Ancylostoma canium), or 700 larvated eggs of whipworm (Trichurisvulpis), or any combination of the three to a healthy canine or feline.Infection was confirmed by microscopic observation of worm ova in fecalsamples obtained from these host animals.

Canine and feline fecal sample preparation. Canine and feline animalsknown to be free of parasitic worm infection or to be infected with one,two or all three of roundworm, hookworm, or whipworm provided the sourceof fecal samples. Samples (approximately 1 gram) from frozen,unpreserved canine or feline fecal samples were suspended in 4 ml ofdiluent solution (“diluent solution” is 0.05 M Tris base; 1 mM EDTA;0.45% Kathon; 16 mg/I gentamicin sulfate; 0.05% Tween-20; 40% fetalbovine serum; 10% rabbit serum; and 5% mouse serum). The suspension wascentrifuged in a tabletop centrifuge at 4000 rpm for 20 minutes toproduce a first supernatant. The first supernatant was centrifuged at10,000 g for 5 minutes to produce a second supernatant, which isreferred to herein as “fecal extract”.

ELISA assays. Purified anti-DIV6728 pAB, anti-DIV6901 pAB andanti-Asp5-1 pAB (100 μl/well; 3 μg/ml for Example 2) were immobilized byphysical adsorption on Immulon 1B 96-well plates overnight at 4° C. Theplates were then blocked with 1% BSA in 0.1M Tris pH 7.0 or 3 hours atroom temperature, followed by 2.5% Sucrose in 0.1 M Tris buffer, pH 7.0for 3 hours at room temperature, aspirating the liquid, drying at roomtemperature. Approximately 100 μl of fecal extract was added to eachwell and allowed to incubate at room temperature for one hour. The wellswere then washed five times with a PBS-Tween-20 solution according tostandard methods known to those of ordinary skill in the art. In aseparate reaction vessel, free anti-DIV6728 pAB, anti-DIV6901 pAB, andanti-Asp5-1 pAB was labeled with horseradish peroxidase (HRP) by usingthe crosslinker succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to create aconjugate, and this conjugate (3 μg/ml for Examples 2) was added to eachwell having immobilized anti-DIV6728 pAB, anti-DIV6901 pAB, andanti-Asp5-1 pAB. Following a 30-minute incubation period at roomtemperature, unbound conjugate was washed from the wells by usingPBS-Tween-20 solution according to standard methods known to those ofordinary skill in the art. 50 μl of TMBLUE® peroxidase substrate(SeraCare Life Sciences, West Bridgewater, Mass.) was then added to eachwell and the plates were incubated for 10 minutes at room temperature.After stopping each enzymatic reaction with 0.1% sodium dodecyl sulfate(SDS) following the 10-minute incubation period, the optical density(OD) value of each well of the 96-well plate was measured at A650 bystandard spectrophotometric techniques by using an ELISA plate reader togenerate an “OD650 value” (or, more simply, an “OD value”) for eachwell. In this arrangement, the OD value obtained for any particular wellof the 96-well plate was directly proportional to the amount ofspecifically bound antigen present in the well (if the IgG is notsaturated).

Example 1

When tested by ELISA in a lateral flow format, anti-DIV6728 pABspecifically binds roundworm coproantigen, anti-DIV6901 pAB specificallybinds whipworm coproantigen, and anti-Asp5-1 pAB specifically bindshookworm coproantigen. There was no cross coproantigen binding betweenthe helminthic antibodies and their non-specific helminthics. Thespecific binding of roundworm coproantigen by anti-DIV6728 pAB, whipwormcoproantigen by anti-DIV6901 pAB, and hookworm coproantigen byanti-Asp5-1 pAB produces a colorimetric change that is readilyobservable to the human eye.

It was a goal of Example 1 to determine whether anti-DIV6728 pAB,anti-DIV6901 pAB, and anti-Asp 5-1 pAB can be used to capture andspecifically bind their respective helminthic coproantigens withoutnon-specific binding in a lateral flow ELISA. The lateral flow formatthat was used was a SNAP® assay device, similar to that which isdescribed in U.S. Pat. No. 5,726,010. Further, the assay was performedgenerally as described in that same patent. Briefly, among othercomponents, the SNAP® assay device included a sample entry cup, a flowmatrix, a sample prefilter pad for removing interfering particulatematter, a specific binding reagent pad, a reactive zone, and anabsorbent reservoir. Anti-DIV6728 pAB, anti-DIV6901 pAB, and anti-Asp5-1 pAB were immobilized in the form of small, round spots at thereactive zone by drying. The reactive zone was then blocked with BSA. Apooled fecal extract (150 μl) from roundworm-infected canines was mixedwith 200 μl (1.0 μg/ml) conjugated anti-DIV6728 pAB, anti-DIV6901 pAB,or anti-Asp 5-1 pAB (the antibodies were affinity-purified before beinglabeled with HRP as described above. This mixture added to the samplecup and then was allowed to flow along the flow matrix. While in theflow matrix, the HRP labeled antibodies specifically bound to theirrespective helminthic coproantigens present in the fecal extract. Theresulting complexes (i.e., those that included the HRP labeledantibodies and their respective helminthic coproantigens) were allowedto specifically bind to the immobilized anti-DIV6728 pAB, anti-DIV6901pAB, or anti-Asp 5-1 pAB at the reaction zone. Flow along the flowmatrix was reversed by contacting the absorbent reservoir with the flowmatrix. At this time, detector and wash solution migrated into the flowmatrix to remove any unbound components and to allow detection of anyanalyte complexes that were present where the capture reagent wasimmobilized onto the reaction zone. (This detection step lasted abouteight minutes.) Stopping of the detection of the analyte complexesoccurred by exposing the analyte complexes to 0.1% sodium azide.

As shown in FIG. 1C-E, detection of helminthic specific analytecomplexes where the roundworm, whipworm, and hookworm specificantibodies were immobilized onto separate reaction zones was visiblyapparent for a roundworm infection (1D), a whipworm infection (1E) and ahookworm infection (1C). In the negative control sample shown in FIG. 1A(diluent solution only), no analyte complexes were detected. In anegative control sample shown in FIG. 1B, no analyte complexes weredetected where the antibody was immobilized onto the reaction zone of aseparate device (the negative control sample was a pool of fecalextracts obtained from canines that did not harbor a helminthinfection). These data therefore indicate that anti-DIV6728 pAB,anti-DIV6901 pAB, and anti-Asp 5-1 pAB can be used in a lateral flowELISA format to separately and specifically bind their respectivehelminthic coproantigen. This specific binding is readily visible to thehuman eye.

Example 2

When tested by ELISA in a microtiter dish format, anti-DIV6728 pABspecifically binds roundworm coproantigen, but does not specificallybind coproantigen from either hookworm, whipworm or heartworm;anti-DIV6901 pAB specifically binds whipworm coproantigen, but does notspecifically bind coproantigen from either hookworm, roundworm orheartworm; and anti-Asp5-1 pAB specifically binds hookworm coproantigen,but does not specifically bind coproantigen from either roundworm,whipworm or heartworm. The specific binding of roundworm coproantigen byanti-DIV6728 pAB, whipworm coproantigen by anti-DIV 6901 pAB, andhookworm coproantigen by anti-Asp5-1 pAB produces a colorimetric changethat is readily observable to the human eye.

It was a goal of Example 2 to determine whether specific binding betweenanti-DIV6728 pAB and roundworm coproantigen; anti-DIV6901 pAB andwhipworm coproantigen; and anti-Asp5-1 pAB and hookworm coproantigen,while the anti-DIV6728 pAB, anti-DIV6901 pAB, and anti-Asp5-1 pAB areimmobilized on a solid support can produce a colorimetric change that isobservable to the human eye.

Referring to FIG. 2, anti-DIV6728 pAB (3 μg/ml) was immobilized onto thebottom surfaces of wells D1-D12 and E1-E12, anti-DIV6901 pAB wasimmobilized on the bottom surfaces of wells G1-G12 and H1-H12, andanti-Asp5-1 pAB was immobilized on the bottom surfaces of wells A1-A12and B1-B12 of a microtiter plate as described before. Following suchimmobilization, the A3, B3, D3, E3, G3 and H3 wells were exposed tofecal extract from a heartworm-infected canine (indicated by “HW” inFIG. 2). The A4, B4, D4, E4, G4 and H4 wells were exposed to fecalextract from a first hookworm-infected canine, the A5, B5, D5, E5, G5,and H5 wells were exposed to fecal extract from a secondhookworm-infected canine, and the A6, B6, D6, E6, G6, and H6 wells wereexposed to fecal extract from a third hookworm-infected canine. The A7,B7, D7, E7, G7 and H7 wells were exposed to fecal extract from a firstroundworm-infected canine, the A8, B8, D8, E8, G8, and H8 wells wereexposed to fecal extract from a second roundworm-infected canine, andthe A9, B9, D9, E9, G9, and H9 wells were exposed to fecal extract froma third roundworm-infected canine. The A10, B10, D10, E 10, G10, and H10wells were exposed to fecal extract from a first whipworm-infectedcanine, the A11, B11, D11, E11, G11, and H11 wells were exposed to fecalextract from a second whipworm-infected canine, and the A12, B12, D12,E12, G12, and H12 wells were exposed to fecal extract from a thirdwhipworm-infected canine. The A1, B1, D1, E1, G1. and H1 wells wereexposed to rDIV6728, rDIV6901, and rAsp5-1(1 μg/ml), and therefore thosewells served as positive controls. The A2, B2, D2, E2, G2, and H2 wellswere not exposed to any fecal extract or to rDIV6728, rDIV6901, andrAsp5-1 and therefore those wells served as negative controls. Afterwashing, wells D1-12 and E1-12 were exposed to HRP-labeled rDIV6728 pAB;wells G1-G12 and H1-H12 were exposed to HRP-labeled rDIV6901 pAB; wellsA1-A12 and B1-B12 were exposed to HRP-labeled rAsp 5-1 pAB at 3 μg/ml asdescribed above.

Following incubation of all of these wells with TMBLUE® peroxidasesubstrate and the subsequent addition of the SDS, colorimetric changewas visually observed in the anti-DIV6728 pAB wells that had beenexposed to fecal extract from roundworm-infected canines (D7-D9 andE7-E9), but no colorimetric change was observed in any of theanti-DIV6728 pAB wells that had been exposed to fecal extract fromcanines infected with either hookworm, whipworm or heartworm.Colorimetric change was visually observed in the anti-DIV6901 pAB wellsthat had been exposed to fecal extract from whipworm-infected canines(G10-G12 and H10-H12), but no colorimetric change was observed in any ofthe anti-DIV6901 pAB wells that had been exposed to fecal extract fromcanines infected with either hookworm, roundworm or heartworm.Colorimetric change was visually observed in the anti-Asp5-1 pAB wellsthat had been exposed to fecal extract from hookworm-infected canines(A4-A6 and B4-B6), but no colorimetric change was observed in any of theanti-Asp5-1 pAB wells that had been exposed to fecal extract fromcanines infected with either roundworm, whipworm or heartworm.

These data indicate that anti-DIV6728 pAB detects roundworm;anti-DIV6901 pAB detects whipworm; and anti-Asp5-1 pAB detects hookwormin an ELISA format sufficiently enough to produce a colorimetric changethat is robust and readily visible to the human eye. Further, these dataindicate that such colorimetric change allows the human eye to readilydistinguish the between the helminthic specific fecal samples containingroundworm, hookworm and whipworm

Example 3

A truncated version of DIV6728, Copro6728, is present in T. canisinfected canine feces.

A. Canine Fecal Sample Preparation

Canine animals known to harbor a roundworm (T. canis) infection or tonot have a parasitic worm infection provided the source of fecalsamples. A sample (approximately 1 gram) of frozen, unpreserved caninefeces pooled from five roundworm-infected or uninfected canines wassuspended in 4 ml of extraction buffer (“extraction buffer” is 1Xphosphate-buffered saline (PBS), pH 7.0-7.5 with 0.05% Tween-20). Thissuspension was vortexed for 2 minutes and then was centrifuged at 13,000rpm for 25 minutes to produce a first supernatant. This firstsupernatant was then centrifuged at 10,000 rpm for 5 minutes to producea second supernatant. This second supernatant hereinafter is referred toas “fecal extract”.

B. Ion Exchange

Ion exchange chromatography can enrich Copro6728 from a fecal sample.PLRS samples were used for this study. Fecal sample was extracted firstwith PBST (0.05% Tween 20), pH 7.3. Sample was diluted with sodiumcitrate buffer, pH 3.0 first and then the pH was adjusted to 3 with HCl.Finally, sample was centrifuged and the supernatant was loaded onto asulfopropyl (SP) column. The SP column was eluted with 20 mM sodiumcitrate buffer, pH 3 with 1 M NaCl, and the elution fractions wereevaluated by ELISA. The ELISA plate was coated with rabbit anti-6728 IgGat 3 μg/ml. Based on the results shown in FIG. 15, it is clear thatCopro6728 can be partially purified and enriched by eluting the SPcolumn with sodium citrate buffer with 1 M NaCl and Copro6728 is in thefraction between A11 and C9 (FIG. 15)

C. Western Blotting and SDS-PAGE

Western blotting and SDS-PAGE gel showed that the molecular weight ofCopro6728 is about 7 kD. Elution fractions from the SP column were mixedand buffer pH was adjusted to 7 with NaOH before loading onto anaffinity column, which was prepared by linking the rabbit anti-6728 IgGwith AminoLink resin (Pierce, Thermo Scientific). The column was washedand eluted according to manufacturer's instructions. Elution fractionswere loaded to a 10 well 4-12% Bis-Tris gradient gel and transferred tonitrocellulose membrane for western blotting. Probed with rabbitanti-6728IgG-HRP, western blotting showed that the major band(Copro6728) is about 7 kD (red arrow on FIG. 16). After furtherconcentration, the same samples were visualized on an SDS-PAGE gel withImperial Protein Stain (Pierce, Thermo Scientific). A 7 kD bandcorresponding to the size indicated by anti-6728IgG-HRP is visible (redarrow on FIG. 17).

D. Mass Spectrometry Analysis

Mass spectrometry analysis on the band cut from SDS-PAGE gel (pointed bya red arrow on FIG. 17) indicated that this band contains Copro6728, andthat the C-terminal portion of DIV6728 contains Copro6728.

The 7 kD band that corresponds to the 7 kD band on the Western blottingwas cut out from the SDS-PAGE gel and sent to the Keck Center at YaleUniversity for Mass spectrometry analysis. The sample in the gel wasfirst trypsin digested and then analyzed by LC-MS/MS using the Q-Tof ofUltima Mass spectrometer (Waters). Two specific peptides were found inthe sample by Mass Spectrometry analysis: Peptide 1: R.FVPCTR.N (SEQ ISNO: 35) and Peptide 2: R.DAEGNCIK.F (SEQ ID NO: 36).

Alignment analysis on the sequences of DIV6728 (SEQ ID NO: 21) and thetwo peptides identified by MS analysis indicated that both peptides arelocated in the C terminal end of the full-length DIV6728, confirmingthat the 7 kb band identified by Western blot is derived from DIV6728.The location of the two peptide sequences indicates that a C-terminalportion of DIV6728 (Copro6728) was present in the T. canis positivefecal samples. FIG. 18 shows the full-length of DIV6728 (SEQ ID NO: 21)with the two peptides identified by MS analysis highlighted in theshaded boxes.

Example 4

Two recombinant proteins were generated that correspond to 64 aminoacids within the N-terminal portion of DIV6728 and 65 amino acids withinthe C-terminal portion of DIV6728.

Based on the MS analysis, western blotting and SDS-PAGE data, two newexpression constructs encoding truncations of DIV6728 were made. Theywere named 6728N (SEQ ID NO: 37) and 6728C (SEQ ID NO: 38) for theN-terminus and C-terminus of full-length DIV6728, respectively. FIG. 19shows an alignment of the 6728N (SEQ ID NO: 37) and 6728C (SEQ ID NO:38) amino acid sequences encoded by the constructs.

A. Synthetic Genes for Expressing Recombinant 6728N and 6728C

The genes for expressing 6728N and 6728C polypeptides were codonoptimized for expression in E. coli, synthesized and cloned into vectorpET28(a) with six His(6) tags at the N-terminus of each recombinantprotein by GeneArt, (Josef-Engert-Str. 11D-93053 Regensburg, Germany).

B. Recombinant Protein 6728N and 6728C Expression

Recombinant proteins 6728N and 6728C were expressed in E. coli BL21(DE3)and purified with a single nickel column. Plasmid pET28(a) 6728N wastransformed into BL21(DE3), grown to an OD˜0.8 and induced with 1 mMIPTG (Isopropyl-1-thio-β-D-galactopyranoside) at 37 ° C. for 2 hour.Cells were lysed with Microfluidizer® Processor, M-11EH. Recombinant6728N was soluble in the 20 mM Tris buffer, pH 8.0, with 500 mM NaCl andwas purified by step eluting the nickel column with differentconcentration of imidazole in the 20 mM Tris buffer, pH 8.0, with 500 mMNaCl. The recombinant 6728N was eluted from the Nickel column by thesame buffer with 500 mM imidazole. FIG. 20 is a SDS-PAGE gel loaded withdifferent samples to check the purification of the recombinant 6728N.Recombinant 6728N is about ˜12 kD in size (lane 9) on the gel.

Plasmid pET28(a) 6728C was transformed into BL21(DE3), grown to an OD˜0.8 and induced with 1 mM IPTG (Isopropyl-1-thio-β-D-galactopyranoside)at 37 ° C. for 2 hour. Cells were lysed with Microfluidizer® Processor,M-11EH. Recombinant 6728C was soluble in the 20 mM Tris buffer, pH 8.0,with 500 mM NaCl and was purified by step eluting the nickel column withdifferent concentration of imidazole in the 20 mM Tris buffer, pH 8.0,with 500 mM NaCl. The recombinant 6728C was eluted from the Nickelcolumn by the same buffer with 500 mM imidazole. FIG. 21 is a SDS-PAGEgel loaded with different samples to confirm the purification of therecombinant 6728C. Recombinant 6728C is about ˜12 kD in size (lane 9) onthis gel.

C. Rabbit Polyclonal Antibodies

Rabbit polyclonal antibody raised against 6728C detects antigen in fecalELISA, whereas polyclonal antibody against 6728N does not detect antigenin fecal ELISA

Recombinant proteins 6728N and 6728C, purified with a single Nickelcolumn, were used to immunize rabbits for polyclonal antibodyproduction. Polyclonal antibodies from the immunized rabbit sera wereaffinity purified with Protein G resin and used to coat Immulon I platesat 2 μg/ml. Four different canine samples were tested with differentantibody coated plates. Antibodies from the two rabbits immunized withrecombinant 6728C could differentiate the T. canis positive fecalsamples from hookworm, whipworm positive samples and nematode negativesamples. However, antibodies from the two rabbits immunized withrecombinant 6728N could not differentiate the T. canis positive fecalsamples from hookworm, whipworm positive samples and nematode negativesamples (FIG. 22). This ELISA data further demonstrates that Copro6728is a C-terminal portion of full-length DIV6728. Further experimentsshowed that antibodies raised against 6728N and 6728C only recognizetheir cognate recombinant proteins without cross reactivity. (FIG. 23).Both of these polyclonal antibodies react with full-length recombinantDIV6728 as expected (FIG. 23).

D. Western Blotting

Rabbit polyclonal antibody against recombinant 6728C can recognize theT. canis positive fecal samples in Western Blotting, but not thepolyclonal antibody against recombinant 6728N. In addition to T. caniswhole worm extract (lane 1), nematode negative (lanes 2-4) and T. canispositive (lanes 5-7) fecal samples were fractionated by SP column withhigh salt elution buffer (1 M NaCl in 20 mM sodium citrate buffer, pH3). The T. canis worm extract (lane 1), samples loaded onto the column(lane 2 and 5), column flow-through (lane 3 and 6), and column elution(lane 4 and 7) were loaded to 10 well, 4-12% Bis-Tris gradient gel andthen further transferred to nitrocellulose membrane, probed withdifferent conjugates as indicated in FIGS. 24-26. Both theanti-full-length 6728 IgG-HRP and anti-6728C-IgG-HRP could differentiatethe T. canis positive fecal sample from the nematode negative fecalsamples (FIGS. 24 and 25). However, the anti-6728N IgG-HRP could notdifferentiate these two different fecal samples (FIG. 26). These datafurther confirmed that Copro6728 is only about half the size offull-length DIV6728, in agreement with the data obtained from Massspectrometry analysis and fecal ELISA.

We claim:
 1. A device for detecting the presence or absence of one ormore helminthic antigens from a sample; the device comprising a solidsupport, wherein the solid support has immobilized thereon at least twoantibodies selected from the group consisting of: (a) a first antibodycapable of specifically binding a roundworm coproantigen, but not awhipworm or hookworm coproantigen; (b) a second antibody capable ofspecifically binding a whipworm coproantigen, but not a roundworm orhookworm coproantigen; and (c) a third antibody capable of specificallybinding a hookworm coproantigen, but not a whipworm or hookwormcoproantigen. 2-61. (canceled)